JP2018537448A - Tissue-specific genome manipulation using CRISPR-Cas9 - Google Patents

Abstract

The present application provides compounds, compositions, their use for the treatment of diseases, conditions, and / or disorders, and their use as asialoglycoprotein receptor (ASGPR) targeting agents.
[Selection] Figure 1

Description

RELATED APPLICATIONS This application is based on US Provisional Patent Application No. 62 / 254,652 filed on November 12, 2015 and US Provisional Patent Application No. 62 / 384,660 filed on September 7, 2016. Claim profit and priority. Each of these provisional patent applications is hereby incorporated by reference in its entirety.

SEQUENCE LISTING This application contains a sequence listing electronically submitted in ASCII format, which is incorporated herein by reference in its entirety. The ASCII copy created on October 7, 2016 is named PCFC-989-WO1_SL. txt, and the size is 8,036,161 bytes.

The present invention relates to compounds, compositions and their use as gene targeting agents.

  Considerable attention has been directed to the development of reagents and methods for delivering bioactive agents to specific tissues, cells, and / or intracellular sites. For example, delivery of large molecules such as antisense or RNAi molecules or proteins is difficult because such compounds are generally unable to permeate cell membranes, where they are often selective for the tissue of interest. , Which in turn increases the risk of off-target pharmacology and raises serious safety concerns. Moreover, selective drug delivery to targeted delivery sites is often difficult because cell permeable molecules are often not selective. One solution for cell diffusion and targeted delivery is conjugation of drugs to targeting agents.

  In gene therapy, most gene editing agents are often delivered via plasmid DNA encapsulated in virus-derived vectors such as adenovirus and adeno-associated virus. Unfortunately, this type of method is a serious problem for patients, mainly i) increased risk of insertion mutations, ii) increased risk of hepatotoxicity due to interaction of viral vectors with Kupffer cells, and iii. ) Problems with only temporary pharmacological effects for patients caused by immunogenic responses to target cells. As a result, there remains a need to find a more effective and safer method for delivering gene editing agents. To date, attempts have been elusive, and in this regard, delivery of gene editing agents in the form of proteins and not in their DNA or RNA represents a tremendous therapeutic opportunity.

  Targeting agents often improve pharmaceutical properties, including pharmacokinetics and pharmacodynamics. The targeting agent allows the drug payload bound to the targeting agent to be efficiently distributed to specific cells and taken up by specific cells. M. in certain sugars such as galactose, N-acetylgalactosamine, and the Journal of the American Chemical Society, 134, 1978 (2012). G. Finn and V.W. Other galactose derivatives, including those described by Mascitti et al., Have been used as targeting agents for hepatocytes because they bind to the asialoglycoprotein receptor (ASGPR) present on the surface of hepatocytes.

  As a further problem with drug delivery, in the mechanism of receptor-dependent endocytosis, the endosomal uptake pathway is known to be a rate-limiting barrier to the delivery of bioactive agents to target intracellular sites. Bioactive molecules are often trapped in endosomal vesicles and degraded in lysosomal compartments. Various reagents, such as chloroquine, polyethyleneimine [PEI], certain polyvalent cation compounds, membrane fusion peptides, and inactivated adenoviruses, rapidly destroy endosomes and deliver bioactive agents in an endosome-like environment. Developed with the goal of minimizing time spent. However, these drugs often lack universality, have a suboptimal ability to promote the cargo's endosomal escape, and are associated with various disadvantages, including toxicity issues.

  Therefore, a versatile and biocompatible cell delivery system would be important for the clinical success of gene targeting and therapeutic drug delivery.

One embodiment of the present invention is a compound of the formula (A-1), (A-2), or (A-3):
Or a pharmaceutically acceptable salt thereof,
Where
R ^xx is, -H, - alkyl, - cycloalkyl, - alkenyl, - alkynyl, - aryl, - ^{heteroaryl, -OR 5, -N (R 4} ) -R 5, a -SR ^5, wherein R ^xx Each of —CH ₂ — groups may be independently replaced with a heteroatom group selected from —O—, —S—, —N (R ⁴ ) —, wherein —CH ₃ of R ^xx is , —N (R ⁴ ) ₂ , —OR ⁴ , and —S (R ⁴ ), which may be replaced with a heteroatom group, the heteroatom group being separated by at least two carbon atoms , The alkyl, cycloalkyl, alkenyl, and alkynyl each independently may be substituted with one or more halo atoms;
R ^yy represents —CN, —CH ₂ —CN, —C≡CH, —CH ₂ —N ₃ , —CH ₂ —NH ₂ , —CH ₂ —N (R ⁴ ) —S (O) ₂ —R ^5. , —CH ₂ —CO ₂ H, —CO ₂ H, —CH ₂ —OH, —CH ₂ —SH, —CH═CH—R ⁵ , —CH ₂ —R ⁵ , —CH ₂ —S—R ⁵ , ^{_{-CH 2 -N (R 4) -R}} 5, -CH 2 -N (R 4) -C (O) -R 5, -CH 2 -N (R 4) -C (O) -O-R 5 , —CH ₂ —N (R ⁴ ) —C (O) —N (R ⁴ ) —R ⁵ , —CH ₂ —O—R ⁵ , —CH ₂ —O—C (O) —R ⁵ , —CH ₂ —O—C (O) —N (R ⁴ ) —R ⁵ , —CH ₂ —O—C (O) —O—R ⁵ , —CH ₂ —S (O) —R ⁵ , —CH ₂ — ^{_{S (O) 2 -R 5,}} -CH 2 -S (O) 2 -N (R 4) -R 5, -C (O) -NH 2, -C (O) -O-R 5, -C (O -N (R ⁴⁾ -R ⁵ or aryl or heteroaryl, said aryl or heteroaryl is optionally substituted with R ^5,
Each R ¹ is independently —CN, —CH ₂ —CN, —C≡CH, —CH ₂ —N ₃ , —CH ₂ —NH ₂ , —CH ₂ —N (R ⁴ ) —S (O ) ₂ —R ⁵ , —CH ₂ —CO ₂ H, —CO ₂ H, —CH ₂ —OH, —CH ₂ —SH, —CH═CH—R ⁵ , —CH ₂ —R ⁵ , —CH ₂ — S—R ⁵ , —CH ₂ —N (R ⁴ ) —R ⁵ , —CH ₂ —N (R ⁴ ) —C (O) —R ⁵ , —CH ₂ —N (R ⁴ ) —C (O) —O—R ⁵ , —CH ₂ —N (R ⁴ ) —C (O) —N (R ⁴ ) —R ⁵ , —CH ₂ —O—R ⁵ , —CH ₂ —O—C (O) — R ⁵ , —CH ₂ —O—C (O) —N (R ⁴ ) —R ⁵ , —CH ₂ —O—C (O) —O—R ⁵ , —CH ₂ —S (O) —R ⁵ , —CH ₂ —S (O) ₂ —R ⁵ , —CH ₂ —S (
^{_{O) 2 -N (R 4)}} -R 5, -C (O) -NH 2, -C (O) -O-R 5, -C (O) -N (R 4) -R 5 , or aryl, Or is heteroaryl, which aryl or heteroaryl is optionally substituted with R ⁵ ,
Alternatively, R ¹ represents —Z—X—Y, —Z—Y, —X—Y, —Z—X ⁺ Y ⁻ , —X ⁺ Y ⁻ , —Z—X ⁻ Y ⁺ , —X ⁻ Y ^+. Or -Y,
X is a linker;
X ⁺ is a positively charged linker;
X ⁻ is a negatively charged linker,
Y is a ribonucleoprotein or endonuclease containing a site-specific modified polypeptide, Y is a site-specific modified polypeptide, Y is a Cas9 ribonucleoprotein, or Y is a Cas9 protein Or Y is a single guide RNA sequence (sgRNA) or Y is a dual guide RNA sequence comprising CRISPR RNA (crRNA) and transactive crRNA (tracrRNA);
Is Y ⁺ a positively charged ribonucleoprotein or endonuclease containing a site-specific modified polypeptide, Y ⁺ is a positively charged site-specific modified polypeptide, or Y ⁺ is positively A charged Cas9 protein,
Y ⁻ is a negatively charged ribonucleoprotein or endonuclease containing a site-specific modified polypeptide, Y ⁻ is a negatively charged site-specific modified polypeptide, or Y ⁻ is negative Either a charged Cas9 ribonucleoprotein, Y ⁻ is a negatively charged sgRNA, or Y ⁻ is a negatively charged dual guide RNA sequence comprising CRISPR RNA (crRNA) and transactive crRNA (tracrRNA) And
Z does not exist or is —C≡C—, —CH═CH—, —CH ₂ —, —CH ₂ —O—, —C (O) —N (R ⁴ ) —, —CH ₂ —. _{S -, - CH 2 -S (} O) -, - CH 2 -S (O) 2 -, - CH 2 -S (O) 2 -N (R 4) -, - C (O) -O-, ^{_{-CH 2 -N (R 4) -}} , - CH 2- N (R 4) -C (O) -, - CH 2 -N (R 4) -S (O) 2 -, - CH 2 -N ( R ⁴ ) —C (O) —O—, —CH ₂ —N (R ⁴ ) —C (O) —N (R ⁴ ) —, —CH ₂ —O—C (O) —, —CH ₂ — O—C (O) —N (R ⁴ ) —, —CH ₂ —O—C (O) —O—, or aryl or heteroaryl, which is optionally substituted with R ⁵ ,
R ² represents —OH, —N ₃ , —N (R ³ ) ₂ , —N (R ³ ) —C (O) —R ³ , —N (R ³ ) —C (O) —N (R ³ ) ₂ , —N (R ³ ) —C (O) —OR ³ , —N (R ³ ) —S (O) ₂ —R ³ , tetrazole, or triazole, which is optionally R Replaced by ³ ,
Each R ³ is independently —H, — (C ₁ -C ₅ ) alkyl, halo-substituted (C ₁ -C ₅ ) alkyl, halo-substituted (C ₃ -C ₆ ) cycloalkyl, (C ₃ -C ₆₎ cycloalkyl, - (C ₁ -C ₅₎ alkenyl, - (C ₁ -C ₅₎ alkynyl, halo-substituted - (C ₁ -C ₅₎ alkenyl or halo-substituted, - at (C ₁ -C ₅₎ alkynyl And the —CH ₂ — group of the alkyl or cycloalkyl may each independently be replaced with a heteroatom group selected from —O—, —S—, and —N (R ⁴ ) — The alkyl —CH ₃ may be each independently replaced with a heteroatom group selected from —N (R ⁴ ) ₂ , —OR ⁴ , and —S (R ⁴ ). Are separated by at least two carbon atoms;
Each R ⁴ is independently —H, — (C ₁ -C ₂₀ ) alkyl, — (C ₁ -C ₂₀ ) alkenyl, — (C ₁ -C ₂₀ ) alkynyl, or (C ₃ -C ₆ ). 1-6 —CH ₂ — groups of the alkyl or cycloalkyl that are cycloalkyl and are separated by at least two carbon atoms are each independently —O—, —S—, or —N (R ⁴⁾ - may be replaced with independently selected heteroatoms from, -CH ₃ of the alkyl are each independently, -N (R ⁴⁾ _2, -OR ^4, and -S (R ⁴ Wherein the heteroatom group is separated by at least two carbon atoms, and the alkyl, alkenyl, alkynyl, and cycloalkyl are substituted with a halo atom. Well,
Each R ⁵ is independently —H, (C ₃ -C ₂₀ ) cycloalkyl, — (C ₁ -C ₆₀ ) alkenyl, — (C ₁ -C ₆₀ ) alkynyl, or (C ₁ -C ₆₀ ). Alkyl, wherein 1 to 6 —CH ₂ — groups of the cycloalkyl or 1 to 20 —CH ₂ — groups of the alkyl are each independently —O—, —S—, and —N; Optionally substituted with a heteroatom independently selected from (R ⁴ ) —, the heteroatom being separated by at least two carbon atoms, each —CH ₃ of the alkyl being independently May be exchanged with a heteroatom group selected from —N (R ⁴ ) ₂ , —OR ⁴ , and —S (R ⁴ ), the heteroatom group being separated by at least two carbon atoms; The alkyl, alkenyl, alkynyl, and cycloalkyl may be substituted with a halo atom.

Another aspect of the present invention is a compound of formula (B):
Or a pharmaceutically acceptable salt thereof,
Where
R ¹ is, -Z-X-Y, -Z -Y, -X-Y, -Z-X + Y -, -X + Y -, -Z-X - Y +, -X - Y +, or -Y,
X is a linker;
X ⁺ is a positively charged linker;
X ⁻ is a negatively charged linker,
Y is a ribonucleoprotein or endonuclease containing a site-specific modified polypeptide, Y is a site-specific modified polypeptide, Y is a Cas9 ribonucleoprotein, or Y is a Cas9 protein Or Y is a single guide RNA sequence (sgRNA) or Y is a dual guide RNA sequence comprising CRISPR RNA (crRNA) and transactive crRNA (tracrRNA);
Is Y ⁺ a positively charged ribonucleoprotein or endonuclease containing a site-specific modified polypeptide, Y ⁺ is a positively charged site-specific modified polypeptide, or Y ⁺ is positively A charged Cas9 protein,
Y ⁻ is a negatively charged ribonucleoprotein or endonuclease containing a site-specific modified polypeptide, Y ⁻ is a negatively charged site-specific modified polypeptide, or Y ⁻ is negative Either a charged Cas9 ribonucleoprotein, Y ⁻ is a negatively charged sgRNA, or Y ⁻ is a negatively charged dual guide RNA sequence comprising CRISPR RNA (crRNA) and transactive crRNA (tracrRNA) And
Z does not exist or is —C≡C—, —CH═CH—, —CH ₂ —, —CH ₂ —O—, —C (O) —N (R ⁴ ) —, —CH ₂ —. _{S -, - CH 2 -S (} O) -, - CH 2 -S (O) 2 -, - CH 2 -S (O) 2 -N (R 4) -, - C (O) -O-, ^{_{-CH 2 -N (R 4) -}} , - CH 2- N (R 4) -C (O) -, - CH 2 -N (R 4) -S (O) 2 -, - CH 2 -N ( R ⁴ ) —C (O) —O—, —CH ₂ —N (R ⁴ ) —C (O) —N (R ⁴ ) —, —CH ₂ —O—C (O) —, —CH ₂ — O—C (O) —N (R ⁴ ) —, —CH ₂ —O—C (O) —O—, or aryl or heteroaryl, which is optionally substituted with R ⁵ ,
R ² represents —OH, —N ₃ , —N (R ³ ) ₂ , —N (R ³ ) —C (O) —R ³ , —N (R ³ ) —C (O) —N (R ³ ) ₂ , —N (R ³ ) —C (O) —OR ³ , —N (R ³ ) —S (O) ₂ —R ³ , tetrazole, or triazole, which is optionally R Replaced by ³ ,
Each R ³ is independently —H, — (C ₁ -C ₅ ) alkyl, halo-substituted (C ₁ -C ₅ ) alkyl, halo-substituted (C ₃ -C ₆ ) cycloalkyl, — (C _1- C ₅₎ alkenyl, - (C ₁ -C ₅₎ alkynyl, halo-substituted - (C ₁ -C ₅₎ alkenyl, halo-substituted - (C ₁ -C ₅₎ alkynyl, or (at C ₃ -C ₆₎ cycloalkyl And the —CH ₂ — group of the alkyl or cycloalkyl may each independently be replaced with a heteroatom group selected from —O—, —S—, and —N (R ⁴ ) — The alkyl —CH ₃ may be each independently replaced with a heteroatom group selected from —N (R ⁴ ) ₂ , —OR ⁴ , and —S (R ⁴ ). Are separated by at least two carbon atoms;
Each R ⁴ is independently —H, — (C ₁ -C ₂₀ ) alkyl, — (C ₁ -C ₂₀ ) alkenyl, — (C ₁ -C ₂₀ ) alkynyl, or (C ₃ -C ₆ ). 1-6 —CH ₂ — groups of the alkyl or cycloalkyl that are cycloalkyl and are separated by at least two carbon atoms are each independently —O—, —S—, or —N (R ⁴⁾ - may be replaced with independently selected heteroatoms from, -CH ₃ of the alkyl are each independently, -N (R ⁴⁾ _2, -OR ^4, and -S (R ⁴ Wherein the heteroatom group is separated by at least two carbon atoms, and the alkyl, alkenyl, alkynyl, and cycloalkyl are substituted with a halo atom. Well,
Each R ⁵ is independently —H, (C ₃ -C ₂₀ ) cycloalkyl, — (C ₁ -C ₆₀ ) alkenyl, — (C ₁ -C ₆₀ ) alkynyl, or (C ₁ -C ₆₀ ). Alkyl, wherein 1 to 6 —CH ₂ — groups of the cycloalkyl or 1 to 20 —CH ₂ — groups of the alkyl are each independently —O—, —S—, and —N; Optionally substituted with a heteroatom independently selected from (R ⁴ ) —, the heteroatom being separated by at least two carbon atoms, each —CH ₃ of the alkyl being independently May be exchanged with a heteroatom group selected from —N (R ⁴ ) ₂ , —OR ⁴ , and —S (R ⁴ ), the heteroatom group being separated by at least two carbon atoms; The alkyl, alkenyl, alkynyl, and cycloalkyl may be substituted with a halo atom.

  Another aspect of the invention includes hereditary angioedema, familial tyrosinemia type I, Alagille syndrome, alpha-1 antitrypsin deficiency, bile acid synthesis and metabolic disorders, biliary atresia, cystic fibrosis liver disease, Idiopathic neonatal hepatitis, mitochondrial hepatic disorder, progressive familial intrahepatic cholestasis, primary sclerosing cholangitis, transthyretin amyloidosis, hemophilia, homozygous familial hypercholesterolemia, familial chylomicron , Hyperlipidemia, steatohepatitis, nonalcoholic steatohepatitis (NASH), nonalcoholic fatty liver disease (NAFLD), hyperglycemia such as type II diabetes, and abnormalities similar to type II diabetes Liver disease or condition in a subject, or liver modulation disease or condition, including but not limited to diseases associated with high liver glucose production For the treatment, the method comprising administering an effective amount of a compound or composition described herein. In some embodiments, the disease or condition is hyperlipidemia, nonalcoholic steatohepatitis (NASH), or nonalcoholic fatty liver disease (NAFLD).

  Another aspect of the invention is a method for selectively modulating transcription of a target DNA in a liver cell of a subject, the DNA comprising hereditary angioedema, familial tyrosine type I, Alagille syndrome, Alpha-1 antitrypsin deficiency, bile acid synthesis and metabolic disorders, biliary atresia, cystic fibrosis liver disease, idiopathic neonatal hepatitis, mitochondrial liver injury, progressive familial intrahepatic cholestasis, primary sclerosis Cholangitis, transthyretin amyloidosis, hemophilia, homozygous familial hypercholesterolemia, familial chylomicronemia, hyperlipidemia, steatohepatitis, nonalcoholic steatohepatitis (NASH), nonalcohol Fatty liver disease (NAFLD), hyperglycemia such as type II diabetes, and diseases with abnormally high liver glucose production similar to type II diabetes But not limited to, liver disease or condition in a subject, or is associated with liver modulated disease or condition, which method comprises administering an effective amount of a compound or composition described herein.

  Another aspect of the invention includes hereditary angioedema, familial tyrosinemia type I, Alagille syndrome, alpha-1 antitrypsin deficiency, bile acid synthesis and metabolic disorders, biliary atresia, cystic fibrosis liver disease, Idiopathic neonatal hepatitis, mitochondrial hepatic disorder, progressive familial intrahepatic cholestasis, primary sclerosing cholangitis, transthyretin amyloidosis, hemophilia, homozygous familial hypercholesterolemia, familial chylomicron , Hyperlipidemia, steatohepatitis, nonalcoholic steatohepatitis (NASH), nonalcoholic fatty liver disease (NAFLD), hyperglycemia such as type II diabetes, and abnormalities similar to type II diabetes Related to a liver disease or condition in a subject, or a liver modulation disease or condition, such as but not limited to Protein for editing a nucleic acid molecule encoding a that includes a method comprising administering an effective amount of a compound or composition described herein.

  Another aspect of the invention includes hereditary angioedema, familial tyrosinemia type I, Alagille syndrome, alpha-1 antitrypsin deficiency, bile acid synthesis and metabolic disorders, biliary atresia, cystic fibrosis liver disease, Idiopathic neonatal hepatitis, mitochondrial hepatic disorder, progressive familial intrahepatic cholestasis, primary sclerosing cholangitis, transthyretin amyloidosis, hemophilia, homozygous familial hypercholesterolemia, familial chylomicron , Hyperlipidemia, steatohepatitis, nonalcoholic steatohepatitis (NASH), nonalcoholic fatty liver disease (NAFLD), hyperglycemia such as type II diabetes, and abnormalities similar to type II diabetes Such as, but not limited to, diseases associated with high liver glucose production Also includes a method comprising administering for regulating the expression level of a gene product, an effective amount of a compound or composition described herein.

  Another aspect of the invention is a composition comprising a ribonucleoprotein described herein (eg, an RNP comprising a site-specific modified polypeptide described herein such as Cas9 RNP or Cpf1 RNP) and an endosome escape agent. Offer things. In some embodiments, the ribonucleoprotein (eg, an RNP comprising a site-specific modified polypeptide as described herein, such as Cas9 RNP or Cpf1 RNP) or the endosomal escape agent is conjugated to an antibody. . In some embodiments, the ribonucleoprotein (eg, an RNP comprising a site-specific modified polypeptide such as Cas9 RNP or Cpf1 RNP described herein) is modified to include a glycosylation site. In some embodiments, the ribonucleoprotein (eg, an RNP comprising a site-specific modified polypeptide as described herein, such as Cas9 RNP or Cpf1 RNP) is modified to include a transduction or translocation domain. Is done.

  Another aspect of the invention provides a composition comprising a Cas9 ribonucleoprotein and an endosomal escape agent. In some embodiments, the Cas9 ribonucleoprotein or the endosomal escape agent is conjugated to an antibody. In some embodiments, the Cas9 ribonucleoprotein is modified to include a glycosylation site. In some embodiments, the Cas9 ribonucleoprotein is modified to include a transduction or translocation domain.

  Another aspect of the invention is (i) a composition comprising a Cas9 ribonucleoprotein and an endosomal escape agent, as described herein, and (ii) a pharmaceutically acceptable excipient, diluent, or A pharmaceutical composition comprising a carrier is included.

  Another aspect of the present invention provides a composition comprising a Cpf1 ribonucleoprotein and an endosomal escape agent. In some embodiments, the Cpf1 ribonucleoprotein or the endosomal escape agent is conjugated to an antibody. In some embodiments, the Cpf1 ribonucleoprotein is modified to include a glycosylation site. In some embodiments, the Cpf1 ribonucleoprotein is modified to include a transduction or translocation domain.

  Another aspect of the invention is a composition comprising (i) a Cpf1 ribonucleoprotein and an endosomal escape agent, as described herein, and (ii) a pharmaceutically acceptable excipient, diluent, or A pharmaceutical composition comprising a carrier is included.

  Another aspect of the invention is a blood disorder, cellular dysregulation or tumor disease and disorder, inflammation and immune related diseases, metabolic, liver, kidney, and protein diseases and disorders, muscle or skeletal diseases, neurological and neurological Ribonucleoproteins as described herein (eg, Cas9 RNP or Cpf1 RNP, etc. herein) for treating diseases and disorders, and diseases or conditions selected from, but not limited to, eye diseases and disorders RNP comprising a site-specific modified polypeptide as described) and a composition comprising administering an endosome escape agent as described herein.

  Another aspect of the invention is a method for selectively modulating transcription of a target DNA of interest, said DNA comprising a blood disorder, cellular dysregulation or tumor disease and disorder, inflammation and immune related diseases, Associated with a disease or condition selected from, but not limited to, metabolic, liver, kidney, and protein diseases and disorders, muscle or skeletal diseases, neurological and neurological diseases and disorders, and eye diseases and disorders; A composition comprising a ribonucleoprotein described herein (eg, an RNP comprising a site-specific modified polypeptide described herein such as Cas9 RNP or Cpf1 RNP) and an endosomal escape agent described herein. A method comprising administering.

  Another aspect of the invention is a blood disorder, cellular dysregulation or tumor disease and disorder, inflammation and immune related diseases, metabolic, liver, kidney, and protein diseases and disorders, muscle or skeletal diseases, neurological and neurological Ribonucleoproteins described herein (eg, Cas9 RNP) for editing nucleic acid molecules encoding proteins associated with diseases and disorders, and diseases or conditions selected from, but not limited to, eye diseases and disorders Or a RNP comprising a site-specific modified polypeptide as described herein, such as Cpf1 RNP), and a composition comprising administering an endosomal escape agent as described herein.

  Another aspect of the invention is a blood disorder, cellular dysregulation or tumor disease and disorder, inflammation and immune related diseases, metabolic, liver, kidney, and protein diseases and disorders, muscle or skeletal diseases, neurological and neurological Ribonucleoproteins described herein (eg, for regulating the expression level of at least one gene product associated with a disease or disorder, and a disease or condition selected from, but not limited to, eye diseases and disorders) A method comprising administering a composition comprising a site-specific modified polypeptide as described herein, such as Cas9 RNP or Cpf1 RNP) and an endosomal escape agent as described herein.

  Another aspect of the invention is a method for site-specific endonucleolytic cleavage of a single stranded RNA (ssRNA) comprising the step of using a compound, RNP, or composition described herein with a protospacer adjacent motif. (PAM) A method comprising administering in the presence of a presenting oligonucleotide (PAMmer) is provided.

  Another aspect of the present invention is a method for site-specific endonucleolytic cleavage of single stranded RNA (ssRNA) comprising the CRISPR / Cas type III-B Cmr complex as described herein. There is provided a method comprising administering a compound or RNP comprising, or a composition thereof.

  It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention as claimed.

FIG. 5 represents the fine crystal structure for compound 23 plotted using an ellipsoid drawn with a confidence level of 50% using the SHELXTL plotting package. 2A-2L represent the results of microscopic image analysis of the RNP construct. FIGS. 2A-2C: Uptake of RNP construct Y53aASGPRL-RNP-EMX1 (A) in HepG2 cells is clearly evident within 60 minutes, as indicated by intracellular points where some surface binding is still evident. The lysosome is labeled with dextran 647 (B) and the nucleus is labeled with Hoechst (C). 2D-2F: The RNP construct Y1C80S-3N-m-RNP-EMX1 (D) shows only cell surface staining at 60 minutes with no evidence of internalization. The lysosome is labeled with dextran 647 (E) and the nucleus is labeled with Hoechst (F). 2G-2I: RNP construct Y53aASGPRL-RNP-EMX1 (G) is completely internalized into HepG2 cells by 20 hours, as indicated by intracellular points. The lysosome is labeled with dextran 647 (H) and the nucleus is labeled with Hoechst (I). 2J-2L: The RNP construct Y53aASGPRL-RNP-EMX1 (J) accumulates on the SK-Hep cell surface within 60 minutes and shows no evidence of internalization. The lysosome is labeled with dextran 647 (K) and the nucleus is labeled with Hoechst (L). Figures 3A and 3B represent mass spectrometry of the Cas9 construct. FIG. 3A represents mass spectrometry of Cas9 construct Y1C80S-3N-m. FIG. 3B represents mass spectrometry of the Cas9 construct Y53Aasgprl. Treated in the presence (co-incubation) of 50 pmol of RNP construct Y53aASGPRL-RNP-EMX1 or Y1C80S-3N-m-RNP-EMX1 and increasing concentrations of ppTG21 endosomal lytic peptides (62.5, 250, 1000, and 2000 nM) FIG. 6 shows the results of editing EMX1 in HepG2 and SkHep cells. RNP alone treatment and lipofection treatment of RNP constructs were included as controls. Represents the results of editing of exons 4 and 5 of PCSK9 in HepG2 and SkHep cells treated in the presence (co-incubation) of 50 pmol RNP construct Y53aASGPRL-RNP-PCS4 and increasing concentrations of ppTG21 endosomal lytic peptides (250 and 1000 nM) . The sequences of exemplary Cas9, Cas9 construct, RNP, RNP construct, and Cpf1 are shown. Continuation of FIG. Continuation of FIG. Continuation of FIG. Continuation of FIG. Continuation of FIG. Continuation of FIGS. 6-6. Continuation of FIGS. 6-7. Continuation of FIGS. 6-8. Continuation of FIG. Continuation of FIG. Continuation of FIG. Continuation of FIG. Continuation of FIG.

DETAILED DESCRIPTION Definitions The present invention can be understood more readily by reference to the following detailed description of exemplary embodiments of the invention and the examples contained therein.

  Unless otherwise defined herein, scientific and technical terms used in this application shall have the meanings that are commonly understood by those of ordinary skill in the art. In general, the chemistry, cell and tissue culture, molecular biology, cell and cancer biology, neurobiology, neurochemistry, virology, immunology, microbiology, pharmacology, genetics, and proteins described herein And terminology used in conjunction with nucleic acid chemistry, and these techniques are well known in the art and commonly used in the art.

  The methods and techniques of the present invention generally follow conventional methods well known in the art, and various general and more specific references cited and discussed throughout this specification, unless otherwise indicated. Done as described. For example, “Principles of Neural Science,” McGraw-Hill Medical, New York, N .; Y. (2000), Motsky, “Intuitive Biostatistics,” Oxford University Press, Inc. (1995), Rodish et al. "Molecular Cell Biology, 4th ed.," W. H. Freeman & Co. , New York (2000), Griffiths et al. "Introduction to Genetic Analysis, 7th ed.", "W. H. Freeman & Co. , N.M. Y. (1999), and Gilbert et al. "Developmental Biology, 6th ed.," Sinauer Associates, Inc. , Sunderland, MA (2000).

  The chemical terms used herein are “The McGraw-Hill Dictionary of Chemical Terms,” Parker S. Ed. McGraw-Hill, San Francisco, C.I. A. (1985), etc., according to conventional usage in the art.

  All publications, patents, and published patent applications mentioned in this application are specifically incorporated herein by reference. In case of conflict, the present specification, including its specific definition, will prevail.

  Throughout this specification, the word “comprise” or variations thereof, eg, “comprises” or “comprising”, is the complete (or component) or complete (or component) mentioned. Is intended to mean the inclusion of the group of, but not any other whole (or component) or exclusion of the whole (or component) group.

  The singular forms “a”, “an”, and “the” include the plural unless the context clearly dictates otherwise.

  The term “including” is used to mean “including but not limited to”. “Including” and “including but not limited to” are used interchangeably.

  Each embodiment of the invention described herein may be used alone or in combination with one or more other embodiments of the invention.

  The term “agent” is used herein to mean a compound (eg, an organic or inorganic compound (eg, a compound of the present invention), a mixture of compounds), a biopolymer (eg, a nucleic acid, an antibody, one of them). , Humanized, chimeric, and human antibodies and monoclonal antibodies, proteins or parts thereof, such as peptides, lipids, carbohydrates) or biological materials such as bacteria, plants, fungi, or animal (especially mammalian) cells or tissues Is used to mean an extract consisting of Agents include, for example, agents that are known with respect to structure and agents that are unknown with respect to structure.

  “Patient”, “subject”, or “individual” are used interchangeably and refer to either a human or non-human animal. These terms include mammals such as humans, primates, livestock animals (including cattle, pigs, etc.), companion animals (eg, dogs, cats, etc.), and rodents (eg, mice and rats). .

  Before the present compounds, compositions, and methods are disclosed and described, it is to be understood that the present invention is not limited to specific synthetic manufacturing methods that can, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting. Plural and singular terms should be treated interchangeably except for displaying numbers.

As used herein, the term “alkyl” refers to a hydrocarbon radical of the general formula C _n H _{2n + 1} . The alkane radical may be linear or branched. For example, the term “(C ₁ -C ₆ ) alkyl” refers to a monovalent straight chain or branched aliphatic group containing 1 to 6 carbon atoms (eg, methyl, ethyl, n-propyl, i-propyl). N-butyl, i-butyl, s-butyl, t-butyl, n-pentyl, 1-methylbutyl, 2-methylbutyl, 3-methylbutyl, neopentyl, 3,3-dimethylpropyl, hexyl, 2-methylpentyl, etc.) Point to. Similarly, the alkyl portion (ie, alkyl portion) of alkoxy, acyl (eg, alkanoyl), alkylamino, dialkylamino, alkylsulfonyl, and alkylthio groups has the same definition as above. When indicated as “optionally substituted”, the alkane radical or alkyl moiety may be unsubstituted or independently selected from the group of substituents set forth below in the definition for “substituted”. It may be substituted with one or more substituents (generally 1-3 substituents except in the case of halogen substituents such as perchloro or perfluoroalkyl). “Halo-substituted alkyl” refers to an alkyl group substituted with one or more halogen atoms (eg, fluoromethyl, difluoromethyl, trifluoromethyl, perfluoroethyl, 1,1-difluoroethyl, etc.).

Similarly, “alkylene” refers to a divalent hydrocarbon radical of the general formula C _n H _2n which may be linear or branched.

  The term “alkenyl” refers to a monounsaturated hydrocarbon radical having one or more carbon-carbon double bonds. The alkenyl moiety may be linear or branched. Exemplary alkenyl groups include ethylenyl. As used herein, “alkenylene” refers to a diunsaturated hydrocarbon radical having one or more carbon-carbon double bonds, which may be linear or branched.

  The term “alkynyl” refers to a monounsaturated hydrocarbon radical having one or more carbon-carbon triple bonds. The alkynyl moiety may be linear or branched.

  As used herein, “alkynylene” refers to a divalent unsaturated hydrocarbon radical having one or more carbon-carbon triple bonds, which may be linear or branched.

  The term “aryl” means a carbocyclic aromatic system containing 1, 2, or 3 rings, which may be fused. When the rings are fused, one of the rings must be fully unsaturated and the fused ring (s) can be fully saturated, partially unsaturated, or completely unsaturated. It may be saturated. The term “fused” means that a second ring exists (ie is bound or formed) by having (ie, sharing) two adjacent atoms in common with the first ring. means. The term “fused” is synonymous with the term “condensed”. The term “aryl” refers to benzyl, phenyl, naphthyl, tetrahydronaphthyl, indanyl, biphenyl, benzo [b] [1,4] oxazine-3 (4H) -onyl, 2,3-dihydro-1H indenyl, and 1, Includes aromatic radicals such as 2,3,4-tetrahydronaphthalenyl.

  The term “cycloalkyl” refers to a non-aromatic ring that is fully hydrogenated and may exist as a monocyclic, bicyclic, or spiro ring. Unless otherwise specified, the carbocycle is generally a 3-20 membered ring. For example, cycloalkyl includes groups such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cyclohexenyl, norbornyl (bicyclo [2.2.1] heptyl), bicyclo [2.2.2] octyl and the like.

  The term “heteroaryl” includes 1, 2, 3, or 4 heteroatoms independently selected from oxygen, nitrogen, and sulfur and has 1, 2, or 3 rings, such Means an aromatic carbocyclic ring system in which the rings may be fused; Condensation is defined above. The term “heteroaryl” includes furyl, thienyl, oxazolyl, thiazolyl, imidazolyl, pyrazolyl, triazolyl, tetrazolyl, isoxazolyl, isothiazolyl, oxadiazolyl, thiadiazolyl, pyridinyl, pyrdiazinyl, pyrimidinyl, pyrazinyl, pyridine-2 (1H) -onyl, Pyridazine-2 (1H) -onyl, pyrimidine-2 (1H) -onyl, pyrazine-2 (1H) -onyl, imidazo [1,2-a] pyridinyl, pyrazolo [1,5-a] pyridinyl, 5,6 , 7,8-tetrahydroisoquinolinyl, 5,6,7,8-tetrahydroquinolinyl, 6,7-dihydro-5H-cyclopenta [b] pyridinyl, 6,7-dihydro-5H-cyclopenta [c] Pyridinyl, 1,4,5,6-tetrahydro Clopenta [c] pyrazolyl, 2,4,5,6-tetrahydrocyclopenta [c] pyrazolyl, 5,6-dihydro-4H-pyrrolo [1,2-b] pyrazolyl, 6,7-dihydro-5H-pyrrolo [ 1,2-b] [1,2,4] triazolyl, 5,6,7,8-tetrahydro- [1,2,4] triazolo [1,5-a] pyridinyl, 4,5,6,7- Examples include, but are not limited to, tetrahydropyrazolo [1,5-a] pyridinyl, 4,5,6,7-tetrahydro-1H-indazolyl, and 4,5,6,7-tetrahydro-2H-indazolyl.

  The term “drug delivery system” refers to a means of delivering a therapeutically effective amount of a ligand, which includes PEG (poly (ethylene glycol) methyl ether), PEG-PLA (poly (ethylene glycol) methyl ether). -Poly (D, L lactide)), PEG-PLGA (poly (ethylene glycol) methyl ether-poly (lactide-co-glycolide)), and PEG-PCL (poly (ethylene glycol) -poly (ε-caprolactone) methyl Ethers), quantum dots (Q dots), liposomes, immunoliposomes, micelles, nanoparticles, and nanogels, but are not limited thereto. Exemplary drug delivery systems are described in Tiwari, G., which is incorporated herein by reference for all purposes. "Drug Delivery Systems: an Updated Review", Int J Pharm Investig 2 (1) p. 2-11 (Jan 2012).

  The term “small molecule” means an organic compound having a molecular weight of 100 to 2,000 daltons, including but not limited to synthetic compounds and natural products.

  An “antibody” is an immunoglobulin molecule capable of specific binding to a target, eg, carbohydrate, polynucleotide, lipid, polypeptide, etc. via at least one antigen recognition site located in the variable region of the immunoglobulin molecule. is there. The term as used herein includes not only intact polyclonal or monoclonal antibodies, but also fragments thereof (eg, Fab, Fab ′, F (ab ′) 2, Fv), single chain (scFv) and domain antibodies. ), As well as fusion proteins comprising an antibody portion, as well as any other modified arrangement of immunoglobulin molecules comprising an antigen recognition site. Antibodies include any class of antibodies, such as IgG, IgA, or IgM (or subclasses thereof), and need not be of a particular class. Depending on the antibody amino acid sequence of the constant domain of its heavy chain, immunoglobulins can be assigned to different classes. There are five major immunoglobulin classes, namely IgA, IgD, IgE, IgG, and IgM, some of which are further subclasses (isotypes), eg, IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. It may be divided into The heavy chain constant domains that correspond to the different classes of immunoglobulins are called alpha, delta, epsilon, gamma, and mu, respectively. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known.

  As used herein, “monoclonal antibody” refers to an antibody obtained from a substantially homogeneous population of antibodies, in other words, the individual antibodies comprising the population may be present in small amounts. , Except for possible spontaneous mutations. Monoclonal antibodies are very specific and are directed to a single antigenic site. Furthermore, in contrast to polyclonal antibody formulations that typically include different antibodies to different determinants (epitopes), each monoclonal antibody is directed to a single determinant on the antigen. The modifier “monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies used in accordance with the present invention may be made by the hybridoma method first described in Kohler and Milstein, 1975, Nature 256: 495, or described in US Pat. No. 4,816,567. Or may be made by recombinant DNA methods. The monoclonal antibodies are also described, for example, in McCafferty et al. , 1990, Nature 348: 552-554.

  The “variable region” of an antibody refers to the variable region of an antibody light chain or the variable region of an antibody heavy chain, either alone or in combination. As is known in the art, the heavy and light chain variable regions each consist of four framework regions (FR) connected by three complementarity determining regions (CDRs), including hypervariable regions. The CDRs of each chain are held in close proximity to the CDRs of the other chain by the FR and contribute to the formation of the antigen binding site of the antibody. Regarding the determination of CDRs, at least two techniques are available: (1) a method based on cross-species sequence variation (ie, Kabat et al. Sequences of Proteins of Immunological Institute, (5th ed., 1991, National Institutes of Health, eHealth, MD)), and (2) methods based on crystallographic studies of antigen-antibody complexes (Al-lazikani et al, 1997, J. Molec. Biol. 273: 927-948). As used herein, a CDR may refer to a CDR defined by either method or a combination of both methods.

  As known in the art, the “constant region” of an antibody refers to the constant region of the antibody light chain or the constant region of the antibody heavy chain, either alone or in combination.

  The expression “therapeutically effective amount” refers to (i) treating a particular disease, condition, or disorder as described herein, (ii) reducing one or more symptoms of a particular disease, condition, or disorder. Means the amount of a compound of the invention that ameliorates, ameliorates or eliminates, or (iii) prevents or delays the onset of one or more symptoms of a particular disease, condition or disorder.

  The term “animal” refers to humans (male or female), companion animals (eg, dogs, cats, and horses), food-source animals, zoo animals, marine animals, birds, and other similar animal species. Point to. “Edible animals” refers to food-source animals such as cows, pigs, sheep, and poultry.

  The expression “pharmaceutically acceptable” means that the substance or composition is chemically and / or toxicologically compatible with the other ingredients that make up the formulation and / or the mammal being treated thereby. Indicates that it must be done.

  The term “treating”, “treat” or “treatment” includes prophylactic, ie, both prophylactic and palliative treatment.

  As used herein, “pharmaceutically acceptable carrier” or “pharmaceutically acceptable excipient” allows an ingredient to maintain biological activity when combined with the active ingredient. And any substance that does not react with the subject's immune system. Examples include, but are not limited to, any of the standard pharmaceutical carriers, for example, phosphate buffered saline, water, emulsions such as oil / water emulsions, and various types of wetting agents. Preferred diluents for aerosol or parenteral administration are phosphate buffered saline (PBS) or physiological (0.9%) saline. Compositions containing such carriers are formulated in well-known conventional methods (eg, Remington's Pharmaceutical Sciences, 18th edition, A. Gennaro, ed., Mack Publishing Co., Easton, PA, 1990, and Remington, The). Science and Practice of Pharmacy, 20th Ed., Mack Publishing, 2000).

  The term “compound of the invention” refers to a compound of any of the formulas described herein, and all their enantiomers, tautomers, and isotopically labeled compounds (unless expressly specified otherwise). Point to. Hydrates and solvates of the compounds of the invention are considered compositions of the invention in which the compound is associated with water or a solvent, respectively. The compounds may also exist in one or more crystalline states, i.e., co-crystals, polymorphs, and they may exist as amorphous solids. All such forms are encompassed by the claims.

  The term “linker” is a chemical group that connects one or more other chemical groups through at least one covalent bond. Examples of the linker include alkylene, alkenylene, alkynylene, alkyl, alkenyl, alkynyl, alkoxy, aryl, heteroaryl, aralkyl, aralkenyl, aralkynyl and the like, and can include one or more spacer groups. The linker may be charge neutral, charge positive or charge negative. In addition, the linker can be cleaved such that the linker can be cleaved or cleaved under certain conditions, connecting the linker to another chemical group within the linker, or the linker's covalent bond attached to a ligand. (E.g., H. Bruyere, et al., “Tuning the pH Sensitives of Orthoester based compounds and Biolative Applications for Biodevelopment of Drugs and Biomedicine 20”). A. Kislukhin et al., “Degradable Conjugates from Oxanorbornadiene Reagents ", Journal of the American Chemical Society, 134, 6491 (2012), which include pH, temperature, salt concentration, catalyst, or enzyme (GM Dubowick, et al. ”Cathepsin B-Labile Dipeptide Linkers for Lysosocial Release of Doxorubicin from Infragenetic Enzymative Energies vity ", Bioconjugate Chemistry, 13, 855 (2002), G. Leriche et al.," Cleaable Linkers in Chemical Biology ", Bioorganic and Medicinal Chemistry, 20, B. , "Chemoselective Ligation and Modification Strategies for Peptides and Proteins", Angewte Chemie International Edition, 47, 10030 (2008), D.C. M.M. Patterson et al. "Finding the Right (Bioorthogonal) Chemistry", ACS Chemical Biology, 9, 592 (2014), C.I. A. Blencove et al. "Self-immobilative Linkers in Polymeric Delivery Systems", Polymer Chemistry, 2, 773 (2011). The disclosures of the above publications are incorporated herein by reference in their entirety for all purposes.

  In some embodiments, the linker is cleavable under intracellular conditions such that cleavage of the linker releases the ligand unit from the compounds of Formulas A and B in the intracellular environment. In some embodiments, the linker is cleavable by a cleaving agent present in the intracellular environment (eg, within a lysosome or endosome or caveolaus). An example of a cleavable linker is a linker that is cleaved enzymatically, ie, a peptidyl linker, which is cleaved by an intracellular peptidase or protease enzyme, including but not limited to lysosomal proteases or endosomal proteases. In some embodiments, the peptidyl linker is at least 2 amino acids long or at least 3 amino acids long. Enzymatic cleavage agents include cathepsins B and D and plasmin, all of which are known to hydrolyze dipeptide drug derivatives resulting in the release of the active drug in the target cell (Dubowchik, Gene M. et al., Catepsin B-Labile Dipeptide Linkers for Lysosomal Release of Doxorubicin, Bioconjugate Chem. 2002, 13, 855-869). Such linkers include, for all purposes, peptides and dipeptides such as those described in the above publications, which are incorporated herein by reference in their entirety.

  Other cleavable linkers can be cleaved by nucleophilic / basic reagents, reducing reagents, light irradiation, and electrophilic / acidic reagents. For example, Leriche, Geoffray, et al. , Cleanable Linkers in Chemical Biology, Bioorganic & Medicinal Chemistry 20 (2012) 571-582.

  In yet another embodiment, the linker unit is not cleavable and the drug is released from the compounds of formula A and B by degradation. This process is often referred to as self-destructive detachment and works by cyclization or electron cascade reactions performed by entropy and thermodynamics. An example of a non-cleavable linker is a polysubstituted electron-rich aromatic species with an amino or hydroxyl group or other electron donating group conjugated to the leaving group at the benzylic position (Blencowe, Christopher A. et. al., Self-immolative Linksers in Polymeric Delivery Systems, Polymer Chemistry, 2011, 2, 773-790). Self-destructing leaving linkers include aniline-based linkers, N-hydroxyaniline-based linkers, phenol-based linkers, 1,8-leaving-based linkers, cyclized-based linkers (that is, hydroxyl-based linkers, amino-based linkers, and thiol-based linkers). Linkers), polymer-dendron conjugates, and polymer conjugates (ie, N- (2-hydroxypropyl) -methacrylamide (HPMA) polymer conjugates, polyethylene glycol (PEG) polymer conjugates). (I. Tranoy-Opalinski, et al., Design of Self-immolative Linkers for Turn-our-Activated Prodrug Therapy, nti-Cancer Agents in Medicinal Chemistry, 2008, 8, 618-637, Blencowe, Christopher A.et al., Self-immolative Linkers in Polymeric Delivery Systems, Polymer Chemistry, 2011, 2, see 773-790).

  Usually, the linker does not substantially cleave in the extracellular environment. As used herein, “not substantially cleaved in the extracellular environment” in the context of a linker means no more than 20% of the linker in a sample of compounds of formulas A and B containing an XY group, Usually less than about 15%, more usually less than about 10%, even more usually less than about 5%, less than about 3%, or less than about 1% of the compound is present in the extracellular environment (eg, plasma) Means to cleave. Whether the linker is substantially cleaved in the extracellular environment can be determined, for example, by incubating the compound with plasma for a predetermined time up to 24 hours (eg, 2, 4, 8, 16, or 24 hours), after which It can be determined by quantifying the amount of free ligand present in the plasma.

The linker may be a monovalent, divalent, or trivalent branched linker. In one embodiment, the linker is a disulfide bridge. In another embodiment, the linker is any of structures L1-L10 that show linkage to Y and Z (Y and Z represent the groups shown in the summary):
Wherein each T is independently absent or is alkylene, alkenylene, or alkynylene, wherein one or more —CH ₂ — groups of the alkylene, alkenylene, or alkynylene are each independently- O -, - S-, and -N (R ⁴⁾ - may be replaced with a heteroatom groups independently selected from the hetero atom group is spaced by at least two carbon atoms, said The alkylene, alkenylene, and alkynylene may each independently be substituted with one or more halo atoms;
Each Q is independently absent or C (O), C (O) —NR ⁴ , NR ⁴ —C (O), O—C (O) —NR ⁴ , NR ⁴ —C (O ) —O, —CH ₂ —, heteroaryl, or a heteroatom group selected from O, S, S—S, S (O), S (O) ₂ , and NR ⁴ , and at least two carbon atoms Separates the heteroatom group O, S, S—S, S (O), S (O) ₂ , and NR ⁴ from any other heteroatom group;
Each R ⁴ is independently —H, — (C ₁ -C ₂₀ ) alkyl, or (C ₃ -C ₆ ) cycloalkyl, wherein the alkyl or cycloalkyl is separated by at least two carbon atoms. 1 to 6 —CH ₂ — groups may be replaced with —O—, —S—, or —N (R ⁴ ) —, and —CH ₃ of the alkyl is —N (R ⁴ ) _2. , —OR ⁴ , and —S (R ⁴ ) may be exchanged with a heteroatom group that is separated by at least two carbon atoms, and the alkyl and cycloalkyl are May be substituted with a halo atom,
Each m is independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40. Each T and each Q in each (TQTQ) is independently selected.

  In one embodiment, Q is heteroaryl selected from 1H-1,2,3-triazolyl, pyridinyl, and 1,2,3,4-tetrazolyl.

  The length of the linker can be adjusted by the value of n to optimize accessibility to the target molecule. In some cases, the optimal length of the linker can be designed by analyzing the space required to properly cleave the drug-target interaction site or compounds of Formulas A and B.

  “Genetically obtained material” refers to proteins, endonucleases, and CRISPR / Cas (eg, Cas9 protein), plasmids (eg, Cas9 protein or Cas9 protein and guide sequences derived from class I, II, or III. Encoding plasmid), RNA sequences such as mRNA, siRNA sequences, and Cas9 ribonucleoprotein are intended.

  As used herein, “site-specific modified polypeptide” refers to CRISPR / Cas endonuclease or derivatives thereof (derived from class I, II, or III). In some embodiments, the site-specific modified polypeptide is a class 2 CRISPR / Cas endonuclease or derivative thereof. In some embodiments, the site-specific modified polypeptide is a Type II CRISPR / Cas endonuclease, eg, Cas9, or a derivative thereof. In some embodiments, the site-specific modified polypeptide is a Type V CRISPR / Cas endonuclease, eg, Cpf1, or a derivative thereof. In some embodiments, the site-specific modified polypeptide is a type III CRISPR / Cas endonuclease. In some embodiments, the site-specific modified polypeptide is a type III-B Cmr complex, eg, a type III-B Cmr complex from Pyrococcus furiosus, Sulfolobus solfarricus, or Thermus thermophilus. For example, Hale, C.I. R. et al. Genes & Development, 2014, 28: 2432-2443, and Makarova K. et al. S. et al. See Nature Reviews Microbiology, 2015, 13, 1-15.

  “Derivative” refers to any polypeptide having an amino acid sequence substantially identical to a naturally occurring polypeptide, wherein one or more amino acids have been modified with side groups of the amino acid. The term “derivative” also refers to a substantial amino acid sequence relative to the native sequence, wherein one or more amino acids have been deleted from, added to, or substituted from the native polypeptide sequence. It also includes any polypeptide that retains homology. Substantial sequence homology is any homology in excess of 50 percent. “Derivatives” are also intended to include fusion Cas9-fluorescent polypeptide fusion proteins such as Cas9 / mcherry, Cas9 / transient domain, Cas9 / endosome escape agent, and Cas9 / NLS. “Derivatives” shall also include fused Cpf1-fluorescent polypeptide fusion proteins such as Cpf1 / mcherry, Cpf1 / transient domain, Cpf1 / endosome escape agent, and Cpf1 / NLS. In addition, “derivative” is also intended to include mutations such as glycosylation site mutations on the polypeptide.

  “Ribonucleoprotein” or “RNP” or “RNP construct” refers to an association that links a ribonucleic acid (RNA) to a protein. In some embodiments, the ribonucleoprotein comprises a site-specific modified polypeptide as described herein. In some embodiments, the ribonucleoprotein comprises a Cas9 protein. In some embodiments, the ribonucleoprotein comprises a Cpf1 protein.

  "Cas9 ribonucleoprotein" or "Cas9 RNP" or "Cas9 RNP construct" is a dual comprising two linked or associated elements: (1) CRISPR RNA (crRNA) and transactive crRNA (tracrRNA) A first element comprising a recognition element comprising either a guide RNA sequence or a single guide RNA sequence (sgRNA), wherein the guide sequence targets sequence specific binding of the Cas9 ribonucleoprotein upon expression And the first element optionally comprising one or more endosomal escape agents, and (2) Cas9 protein and optionally one or more nuclear localization sequences (NLS) and optionally one Cas9 constructs comprising the above fluorescent proteins, as well as one or more endosaws Comprises a second element including an escape agent, said first element is associated to said second element. Where a compound of formula A-1, A-2, A-3, or B is included, the compound is conjugated to either the first or second element of the RNP construct.

  (1) a first element comprising a recognition element comprising a guide sequence, which guide sequence directs the sequence-specific binding of Cpf1 ribonucleoprotein to a target sequence upon expression, and optionally 1 The first element comprising one or more endosomal escape agents, and (2) a Cpf1 construct comprising a Cpf1 protein and optionally one or more nuclear localization sequences (NLS) and optionally one or more fluorescent proteins, and 1 A “Cpf1 ribonucleoprotein” comprising a second element comprising one or more endosomal escape agents, wherein the first element is associated with the second element. Where a compound of formula A-1, A-2, A-3, or B is included, the compound is conjugated to either the first or second element of the RNP construct.

  In some embodiments of ribonucleoprotein, the guide sequence directs the sequence specific binding of the ribonucleoprotein to a target sequence in the cell. The cell may be eukaryotic or prokaryotic. In some embodiments, the second element comprises about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 NLS, amino terminus, carboxy terminus, or combinations thereof (Eg, one or more NLS at the amino terminus and one or more NLS at the carboxy terminus). Where there are multiple NLSs, each may be selected independently of the other, so a single NLS may be in multiple copies and / or one or more other included in one or more copies. May be included in combination with NLS.

  A “guide RNA sequence” is one whose complementarity to a target polynucleotide sequence is sufficient to hybridize to the target sequence and to direct sequence specific binding of a ribonucleoprotein described herein to the target sequence. Any polynucleotide sequence. In some embodiments, a “guide RNA sequence” comprises (i) a moiety that is sufficient to hybridize to the target sequence and (ii) a site-specific modified protein. Any polynucleotide sequence that has a portion that binds (eg, Cas9, cpf1, etc. as described herein) and directs it to the target sequence. For example, Jinek et al. , Science 2012, Briner et al. Mol. See Cell 2014. Exemplary Cas9 proteins, Cas9 guide RNA sequences, plasmids, and ribonucleoproteins are published in US20140068797 published on March 6, 2014, US201403134 published on 01/29/2015, and 3/19/2015. US2015056881. All of these publications are incorporated herein in their entirety for all purposes.

  In some embodiments, the guide arrangement is at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20 , At least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, or at least 30 nucleotides, and complementarity between the guide sequence and its corresponding target sequence Degrees are approximately 50%, 60%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90% when optimally aligned using an appropriate alignment algorithm. 91%, 92%, 93%, 94%, 95 96%, 97%, 98%, 99%, or more, or about 50%, 60%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, More than 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more. Optimal alignment can be determined using any suitable algorithm to align the sequences. Non-limiting examples include Smith-Waterman algorithm, Needleman-Wunsch algorithm, Burrows-Wheeler Transformer (e.g. Burrows Weeler Aligner), ClustalW, Clustal X, BLAT, NovoLig, San Diego, California), SOAP (available at soap.genomics.org.cn), and Mac (available at maq.sourceforge.net).

  The Cas9 protein is aureus, S .; pneumoniae, S .; pyogenes, S .; thermophilus, N.M. meningitidis, or A. It may be derived from ebreus. In some embodiments, the Cas9 protein exhibits a conserved structure having an HNH homing endonuclease domain and a split RuvC / RNaseH endonuclease domain, each Cas9 protein being four major motifs, namely a RuvC-like motif It shares motifs 1, 2, and 4 and motif 3, which is an HNH motif. With respect to Streptococcus pyogenes (SEQ ID NO: 8), motif 1 is SEQ ID NO: 260, motif 2 is SEQ ID NO: 261, motif 3 is SEQ ID NO: 262, and motif 4 is SEQ ID NO: 263. Thus, “Cas9 protein sequence” or “Cas9 protein” means a polypeptide comprising an amino acid sequence having at least four motifs within the sequence, wherein the motif is any Cas9 amino acid of any of SEQ ID NOs: 260-263. At least about 75 percent, at least about 80 percent, at least about 85 percent, at least relative to the corresponding portion of any of the amino acid sequences shown in SEQ ID NOS: 1, 2, 3, and 4 or SEQ ID NOS: 1-829 It has about 90 percent, at least about 95 percent, at least about 96 percent, at least about 97 percent, at least about 98 percent, at least about 99 percent, or 100 percent amino acid sequence identity. In another embodiment, the Cas9 amino acid sequence is at least about 75 relative to the corresponding amino acids of amino acid positions 7-166, or 731-1003, or SEQ ID NOs: 1-7, 9-829 of SEQ ID NO: 8. Percent, at least about 80 percent, at least about 85 percent, at least about 90 percent, at least about 95 percent, at least about 96 percent, at least about 97 percent, at least about 98 percent, at least about 99 percent, or 100 percent amino acid sequence identity It is.

  In some embodiments, the Cas9 protein is S. cerevisiae. pyogenes Cas9 (wild type) (SEQ ID NO: 848), S. pyogenes Cas9 mutant M1C (SEQ ID NO: 849), S. cerevisiae. pyogenes Cas9 mutants M1C and C80S (SEQ ID NO: 850), S. cerevisiae. pyogenes Cas9 nickase mutant D10A (SEQ ID NO: 851), S. cerevisiae. pyogenes Cas9 nickase mutant H840A (SEQ ID NO: 852), S. pyogenes Cas9 nickase variants E923P and T924P (SEQ ID NO: 853), Acidovorax ebreus Cas9 (SEQ ID NO: 854), acidic mine drainage bacteria Ga0052161_JGI Cas9 (SEQ ID NO: 855), S. selected from pyogenes Cas9 null mutants D10A and H840A (SEQ ID NO: 1027), and the uranium mine bacterium FW106_JGI Cas9 (SEQ ID NO: 856).

  Cpf1 protein is A.I. sp. , L. bacterium, p. macacae, and P.I. may be from disiens. In some embodiments, the Cpf1 protein is A.I. sp Cpf1 (SEQ ID NO: 857), L. Bacterium Cpf1 (SEQ ID NO: 858), P.I. macacae Cpf1 (SEQ ID NO: 859), P. a. selected from disiens Cpf1 (SEQ ID NO: 860), but is not limited thereto. In some embodiments, the Cpf1 protein is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least with respect to any one of SEQ ID NOs: 857-860. An amino acid sequence having 99% or 100% amino acid sequence identity can be included.

  “Nuclear localization sequence” (NLS) refers to an amino acid sequence that helps a ribonucleoprotein of the invention, eg, the Cas-9 ribonucleoprotein, enter the nucleus of a eukaryotic cell. Thus, NLS typically contains one or more short sequences of positively charged lysine or arginine exposed on the protein surface. Exemplary NLS include the NLS of the SV40 viral large T antigen having the amino acid sequence PKKKRKV (SEQ ID NO: 830), the NLS derived from nucleoplasmin (eg, the nucleoplasmin bisection NLS of the sequence KRPAATKAGQAKKKK (SEQ ID NO: 831), the amino acid sequence PAAKRVKLD RNV of RBRI derived from RNRI with RKRI from RKRI, which is an RNRI of RKRI from RNRI with the sequence of RNRI having the sequence of NRRI having the sequence of RQR with the sequence of QR ) (SEQ ID NO: 8 5), myoma T protein sequences VSRKRPRP (SEQ ID NO: 836) and PPKKARED (SEQ ID NO: 837), human p53 sequence PQPKKKKPL (SEQ ID NO: 838), mouse c-abl IV sequence SALIKKKKKKMAP (SEQ ID NO: 839), influenza virus NS1 Of sequences DRLRR (SEQ ID NO: 840) and PKQKKRK (SEQ ID NO: 841), hepatitis delta virus antigen sequence RKLKKKIKKL (SEQ ID NO: 842), mouse Mx1 protein sequence REKKKKLKRR (SEQ ID NO: 843), human poly (ADP-ribose) polymerase Sequence KRKGDEVDGVDEVAKKKSKK (SEQ ID NO: 844), steroid hormone receptor (human) glucocorticoid sequence RKCLQAGMNLEARKTKK (sequence No. 845), the sequence MAPKKKRKVGIHRGVP (SEQ ID NO: 1035), and the NLS sequence derived from the sequence PKKKRKVEDPKKKKRKVD (SEQ ID NO: 1036).

  As used herein, "Cas9 construct Y1C80S-3N-m" or "Y1C80S-3N-m" pyogenes Cas9 mutants M1C and C80S (amino acid sequence) -3NLS and mCherry (SEQ ID NO: 1015).

  As used herein, "Cas9 construct Y1C80S-2N" or "Y1C80S-2N" pyogenes Cas9 mutant M1C and C80S (amino acid sequence) -2NLS (SEQ ID NO: 1013).

As used herein, “Cas9 construct Y53aASGPRL” or “Y53aASGPRL” can be obtained at cysteine positions 1 and 574 (addition of 2 × 2165 Da) via formation of a disulfide bond with the S atom of cysteine. Labeled with two copies (compound 53 used as reactant, see experimental section for further details) pyogenes Cas9 variants M1C and C80S (amino acid sequence) -3NLS and mCherry (SEQ ID NO: 1015) refer to:

  As used herein, “RNP construct Y53aASGPRL-RNP-EMX1” or “Y53aASGPRL-RNP-EMX1” is a molar ratio of (1) Cas9 construct Y53aASGPRL and (2) EMX1 single guide sgRNA sequence (SEQ ID NO: 907). Refers to RNP constructs prepared by co-incubation at 1: 1.2.

  As used herein, “RNP construct Y1C80S-3N-m-RNP-EMX1” or “Y1C80S-3N-m-RNP-EMX1” refers to (1) Cas9 construct Y1C80S-3N-m and (2) EMX1. Refers to the RNP construct prepared by co-incubation of a single guide sgRNA sequence (SEQ ID NO: 907) at a molar ratio of 1: 1.2.

  As used herein, “RNP construct Y53aASGPRL-RNP-PCS4” or “Y53aASGPRL-RNP-PCS4” refers to (1) Cas9 construct Y53aASGPRL and (2) exon 4 and 5 loci of PCSK9 (SEQ ID NO: 906). ) Refers to the RNP construct prepared by co-incubation at a molar ratio of sgRNA of 1: 1.2.

  As used herein, “RNP construct Y53aASGPRL-RNP-PCS1” or “Y53aASGPRL-RNP-PCS1” refers to (1) the sgRNA targeting the Cas9 construct Y53aASGPRL and (2) the PCSK9 gene (SEQ ID NO: 896). Refers to an RNP construct prepared by co-incubation at a molar ratio of 1: 1.2.

  As used herein, an “endosomal escape agent” refers to an agent that facilitates an agent, such as a compound, DNA, siRNA, polypeptide, or ribonucleoprotein, to escape the endosome of a cell. Endosome escape agents for use in the present invention include, but are not limited to, lysosome agonists, cell permeable peptides, membrane fusion peptides, endosome lytic peptides, pore formers, and proton sponge agents. The lysosome agonist is a weak base and can penetrate the lysosome as a protonated form, raising the intracellular pH. Cell penetrating peptides (CPPs) are a diverse class of peptides that typically comprise 5 to 30 amino acids and can penetrate cell membranes. Membrane fusion peptides are short peptides that destabilize phospholipid membranes. A pore-forming agent is an agent such as a peptide that induces pore formation through the membrane and thereby destroys the endosome. A proton sponge agent is an agent having a plurality of proton acceptor sites that destroy endosomes by osmolytic action. An endosomal lytic peptide can be a polyanionic peptide or peptidomimetic that exhibits pH-dependent membrane lytic activity and promotes endosomal lysis or leakage, or a peptide with a neutral or near neutral charge at physiological pH . In certain embodiments, the endosomal lytic peptide assumes its active conformation at the endosomal pH. The “active” conformation is a conformation in which the endosomal lytic component promotes endosomal lysis and / or transport of the modular composition of the invention, or component thereof, from the endosome to the cytoplasm of the cell.

  In some embodiments, the endosomal escape agent is associated with an RNP construct. In other embodiments, the endosomal escape agent is conjugated to the first or second element of the RNP construct. In further embodiments, the endosomal escape agent is conjugated to a compound of formula A-1, A-2, A-3, or B.

Compound One embodiment of the present invention is the compound of formula (A-1), (A-2), or (A-3):
Or a pharmaceutically acceptable salt thereof.

In some embodiments of the compound of formula (A-1), (A-2), or (A-3), the site-specific modified polypeptide is a Cas9 protein or a Cpf1 protein. In some embodiments of the compound of formula (A-1), (A-2), or (A-3), Y is a Cas9 ribonucleoprotein, a Cas9 protein, a single guide RNA sequence (sgRNA), or a CRISPR A dual guide RNA sequence comprising RNA (crRNA) and transactive crRNA (tracrRNA), Y ⁺ is a positively charged Cas9 protein, Y ⁻ is a negatively charged Cas9 ribonucleoprotein, negatively charged Or a negatively charged dual guide RNA sequence comprising CRISPR RNA (crRNA) and transactive crRNA (tracrRNA).

Another aspect of the invention is a compound of formula (B) as described in the overview:
Or a pharmaceutically acceptable salt thereof.

In some embodiments of the compound of formula (B), the site-specific modified polypeptide is a Cas9 protein or a Cpf1 protein. In some embodiments of the compound of formula (B), Y comprises a Cas9 ribonucleoprotein, a Cas9 protein, a single guide RNA sequence (sgRNA), or a dual comprising CRISPR RNA (crRNA) and a transactive crRNA (tracrRNA). Guide RNA sequence, Y ⁺ is a positively charged Cas9 protein, Y ⁻ is a negatively charged Cas9 ribonucleoprotein, negatively charged sgRNA, or CRISPR RNA (crRNA) and transactive crRNA ( A negatively charged dual guide RNA sequence comprising tracrRNA).

In some embodiments of the compound of formula (A-1), (A-2), (A-3), or (B), R ² is —NH—C (O) —CH ₃ .

Formula (A-1), in some embodiments of the compounds of (A-2), (A -3), or (B), R ¹ is, -Z-X ⁺ Y ^- it is. In some embodiments, R ¹ is —ZX ⁻ Y ⁺ . In some embodiments, R ¹ is —Z—X—Y. In some embodiments, R ¹ is —Z—X—Y and is selected from the group consisting of L1-L10:
Here, each T is independently absent or is (C ₁ -C ₁₀ ) alkylene, (C ₂ -C ₁₀ ) alkenylene, or (C ₂ -C ₁₀ ) alkynylene, One or more carbon groups may each independently be replaced with a heteroatom group independently selected from —O—, —S—, and —N (R ⁴ ) —, Separated by at least two carbon atoms, the alkylene, alkenylene, and alkynylene may each independently be substituted with one or more halo atoms;
Each Q is independently absent or C (O), C (O) —NR ⁴ , NR ⁴ —C (O), O—C (O) —NR ⁴ , NR ⁴ —C (O ) —O, —CH ₂ —, heteroaryl, or a heteroatom group selected from O, S, S—S, S (O), S (O) ₂ , and NR ⁴ , and at least two carbon atoms Separates the heteroatom group O, S, S—S, S (O), S (O) ₂ , and NR ⁴ from any other heteroatom group;
Each R ⁴ is independently —H, — (C ₁ -C ₂₀ ) alkyl, — (C ₁ -C ₂₀ ) alkenyl, — (C ₁ -C ₂₀ ) alkynyl, or (C ₃ -C ₆ ). 1-6 —CH ₂ — groups of the alkyl or cycloalkyl that are cycloalkyl and separated by at least two carbon atoms are replaced with —O—, —S—, or —N (R ⁴ ) —. And the —CH ₃ of the alkyl may be replaced with a heteroatom group selected from —N (R ⁴ ) ₂ , —OR ⁴ , and —S (R ⁴ ). Are separated by at least two carbon atoms, the alkyl, alkenyl, alkynyl, and cycloalkyl may be substituted with a halo atom;
Each m is independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40. In some embodiments, Q is heteroaryl independently selected from 1H-1,2,3-triazolyl, pyridinyl, and 1,2,3,4-tetrazolyl.

In some embodiments of the compound of formula (A-1), (A-2), (A-3), or (B), X, X ⁺ , or X ⁻ comprises a disulfide bond.

Another embodiment of the present invention is a compound of formula (C-1), (C-2), (C-3), or (C4):
Or a pharmaceutically acceptable salt thereof,
Where
n is 1, 2 or 3,
W is absent or is a peptide,
L is-(TQ-TQ) _m- ,
Each T is independently absent or (C ₁ -C ₁₀ ) alkylene, (C ₂ -C ₁₀ ) alkenylene, or (C ₂ -C ₁₀ ) alkynylene, wherein one or more of the T The carbon groups may each independently be replaced with a heteroatom group independently selected from —O—, —S—, and —N (R ⁴ ) —, wherein the heteroatom group is at least 2 Separated by two carbon atoms, the alkylene, alkenylene, and alkynylene may each independently be substituted with one or more halo atoms;
Each Q is independently absent or C (O), C (O) —NR ⁴ , NR ⁴ —C (O), O—C (O) —NR ⁴ , NR ⁴ —C (O ) —O, —CH ₂ —, heteroaryl, or a heteroatom group selected from O, S, S—S, S (O), S (O) ₂ , and NR ⁴ , and at least two carbon atoms Separates the heteroatom group O, S, S—S, S (O), S (O) ₂ , and NR ⁴ from any other heteroatom group;
Each R ⁴ is independently —H, — (C ₁ -C ₂₀ ) alkyl, — (C ₁ -C ₂₀ ) alkenyl, — (C ₁ -C ₂₀ ) alkynyl, or (C ₃ -C ₆ ). 1-6 —CH ₂ — groups of the alkyl or cycloalkyl that are cycloalkyl and separated by at least two carbon atoms are replaced with —O—, —S—, or —N (R ⁴ ) —. And —CH ₃ of the alkyl may be replaced with a heteroatom group selected from —N (R ⁴ ) 2, —OR ⁴ , and —S (R ⁴ ), and the heteroatom group Are separated by at least two carbon atoms, the alkyl, alkenyl, alkynyl, and cycloalkyl may be substituted with a halo atom;
Each m is independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40,
Other parts are as defined in formula (A-1), (A-2), or (A-3).

Another embodiment of the present invention is a compound of formula (D-1) or (D-2):
Or a pharmaceutically acceptable salt thereof,
In the formula, Y is as defined in formula (A-1), (A-2), or (A-3).

Another aspect of the present invention is a compound of formula (E):
Or a pharmaceutically acceptable salt thereof,
Where
n is 1, 2 or 3,
W is absent or is a peptide,
L is − (TQTQ) _m ,
Each T is independently absent or (C ₁ -C ₁₀ ) alkylene, (C ₂ -C ₁₀ ) alkenylene, or (C ₂ -C ₁₀ ) alkynylene, wherein one or more of the T The carbon groups may each independently be replaced with a heteroatom group independently selected from —O—, —S—, and —N (R ⁴ ) —, wherein the heteroatom group is at least 2 Separated by one carbon atom, the alkylene, alkenylene, alkynylene may each independently be substituted with one or more halo atoms;
Each Q is independently absent or C (O), C (O) —NR ⁴ , NR ⁴ —C (O), O—C (O) —NR ⁴ , NR ⁴ —C (O ) —O, —CH ₂ —, heteroaryl, or a heteroatom group selected from O, S, S—S, S (O), S (O) ₂ , and NR ⁴ , and at least two carbon atoms Separates the heteroatom group O, S, S—S, S (O), S (O) ₂ , and NR ⁴ from any other heteroatom group;
Each R ⁴ is independently —H, — (C ₁ -C ₂₀ ) alkyl, — (C ₁ -C ₂₀ ) alkenyl, — (C ₁ -C ₂₀ ) alkynyl, or (C ₃ -C ₆ ). 1-6 —CH ₂ — groups of the alkyl or cycloalkyl that are cycloalkyl and separated by at least two carbon atoms are replaced with —O—, —S—, or —N (R ⁴ ) —. And the —CH ₃ of the alkyl may be replaced with a heteroatom group selected from —N (R ⁴ ) ₂ , —OR ⁴ , and —S (R ⁴ ). Are separated by at least two carbon atoms, the alkyl, alkenyl, alkynyl, and cycloalkyl may be substituted with a halo atom;
Each m is independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40,
The other part is as defined by the formula (B).

Another embodiment of the present invention is a compound of formula (F-1) or (F-2):
Or a pharmaceutically acceptable salt thereof,
Y is as defined in Formula (B).

  Formula (C-1), (C-2), (C-3), (C4), (D-1), (D-2), (E), (F-1), or (F-2) In some embodiments of the compound of), n is 1. In some embodiments, n is 2. In some embodiments, n is 3.

In some embodiments of the compound of formula (E), the compound:
Or
Or a pharmaceutically acceptable salt thereof.

  In some embodiments of the compounds of the invention, W is absent. In some embodiments, W is a peptide comprising an endosomal lytic peptide or a nuclear localization peptide. In some embodiments, the endosomal lytic peptide exhibits pH dependent membrane lytic activity, a polyanionic peptidomimetic that results in endosomal lysis or leakage, or a neutral or near neutral charge at physiological pH. It may be a peptide having. In certain embodiments, the endosomal lytic peptide assumes its active conformation at endosomal pH. The “active” conformation is a conformation in which the endosomal lytic component promotes endosomal lysis and / or transport of the modular composition of the invention, or component thereof, from the endosome to the cytoplasm of the cell. For example, Martin M.M. E. et al. See Peptide-guided gene delivery, the AAPS Journal, 2007, 9 (1), article 3.

In some embodiments, W is selected from, but not limited to, CHK ₆ HC (SEQ ID NO: 861), H5WYG (SEQ ID NO: 1029), GLFHAIAHFIHGGWHGLIHGGWYG (SEQ ID NO: 862), and derivatives thereof. It is a peptide containing a peptide.

  In some embodiments, W is an influenza HA-2 peptide, ppTG21 (SEQ ID NO: 1012), Aurein 1.2, GLFDIIKKIAESF (SEQ ID NO: 1023), GLFGAIAGFIENGWEGMIDGWYG (SEQ ID NO: 863), Melittin, GIGAVLKVLTTGLLPLIQQ , Tat (48-60), GRKKRRQRRRPPPQ (SEQ ID NO: 865), penetratin, RQIKIWFQNRRMKWKK (SEQ ID NO: 866), transportan, GWTLNSAGYLLLGKINLKALAALAKIL (SEQ ID NO: 867), GALA peptide, WALALA sequence: WELALA KALAKALAKHLAKALAKALKACEA (SEQ ID NO: 869), JST-1, GLFALLELLLESLWELLEA (SEQ ID NO: 870), ppTG1, GLFKALLKLLKSLWKLLLKA (SEQ ID NO: 871), ppTG20, GLFRRLL A peptide comprising a fusion peptide.

In some embodiments, W is a proteasome peptide with a Gly-Ala repeat, eg, a peptide comprising CWK ₁₈ (GA) ₄ (SEQ ID NO: 873).

  In some embodiments, W is an SV40 T antigen, PKKKRKV (SEQ ID NO: 830), SV40 Vp3, KKKRK (SEQ ID NO: 874), adenovirus E1a, KRPRP (SEQ ID NO: 875), human c-myc, PAAKRVKLD (SEQ ID NO: 832), RQRRNELKRSP (SEQ ID NO: 833), and peptides comprising single segment NLS, including but not limited to derivatives thereof. In some embodiments, the peptide is a nucleoplasmin, KRPAATKKAGQAKKKK (SEQ ID NO: 831), Xenopus Nl, VRKKRKTEEEESPLKDKAKSKKQE (SEQ ID NO: 876), mouse FGF3, RLRRDGRGRGKKR8K And bi-segment NLS including, but not limited to, and derivatives thereof. In some embodiments, the peptide comprises a non-classical NLS, eg, the M9 peptide, NQSSNFGPMGGGNFGSGSGPYGGGGQYFAKPRNQGGY (SEQ ID NO: 834).

  In some embodiments, W is RGD peptide, ICRRARGDNPDDRCT (SEQ ID NO: 1037), integrin binding peptide, PLAIDIDGIELTY (SEQ ID NO: 1038), secretin, HSDGTFTSELSRLRDSARQRLLLQGLV (SEQ ID NO: 879), GE7 (EGFIGIG sequence), NPVPVGY 880), neurotensin, ELYENKPRRPYIL (SEQ ID NO: 881), LOX-1 binding peptide, LSIPPKA (SEQ ID NO: 882), FQPTPQL (SEQ ID NO: 883), LTPATAI (SEQ ID NO: 884), or derivatives thereof Peptides including but not limited to cell targeting peptides.

  In some embodiments of the compounds of the invention, Y is a Cas9 ribonucleoprotein. In some embodiments, Y comprises a recognition element comprising either (1) a dual guide RNA sequence comprising CRISPR RNA (crRNA) and transactive crRNA (tracrRNA), or a single guide RNA sequence (sgRNA). A first element, wherein the guide sequence directs sequence-specific binding of the Cas9 ribonucleoprotein to a target sequence upon expression, and (2) the Cas9 protein and optionally A Cas9 ribonucleoprotein comprising a second element comprising one or more nuclear localization sequences (NLS), wherein the first element is associated with the second element. In some embodiments, the target sequence is a eukaryotic target sequence. In another embodiment, the target sequence is a prokaryotic target sequence.

  In some embodiments, as used herein, the tracrRNA, the crRNA, or the sgRNA may each be optionally independently chemically modified. Suitable chemical modifications include backbone modifications, base modifications, and sugar modifications. Exemplary chemical modifications include the use of phosphorothioate linkages (eg, phosphorothioate (PS) linkages can be made by replacing one non-bridging oxygen atom on the backbone phosphate between two ribonucleotides with a sulfur atom), boranoline Use of acid bonds (eg creation of boron-phosphorus bonds by introduction of boron atoms in place of the non-bridging oxygen atoms), use of locked nucleic acid (LNA) nucleotides containing a methylene bridge between the 2 ′ and 4 ′ carbons of the ribose ring Chemical modification at the 2 ′ position of the ribose sugar (eg, the use of RNA analogs is 2′-O-methyl RNA, 2′-O-methoxyethyl (2′-MOE) RNA, and 2′-fluoro RNA Modification of the 4′-thio position by introducing a sulfur atom in place of the oxygen bonded to the 4 ′ carbon of the ribose ring, ribodifluoro Introduction of Ruiru (rF) nucleotides, and the introduction of PNA or morpholino monomers but are not limited to.

  In some embodiments, the first element of the Cas9 ribonucleoprotein comprises a dual guide RNA sequence comprising CRISPR RNA (crRNA) and transactive crRNA (tracrRNA), the remainder of the compound comprising the Cas9 One or more of each is linked to a ribonucleoprotein by an independent interaction with the tracrRNA or the crRNA, and the tracrRNA or the crRNA may optionally be chemically modified. In some embodiments, the tracrRNA and the crRNA are combined to form a dual RNA guide. In some embodiments, the tracrRNA-crRNA dual RNA guide is at least 50 when the degree of complementarity between the dual RNA guide sequence and its corresponding target sequence is optimally aligned using a suitable alignment algorithm. %, 60%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%.

  In some embodiments of the Cas9 ribonucleoprotein, the tracrRNA comprises at least about 75 percent, at least about 80 percent, at least about 80 percent, at least about 80 percent, at least about 80 percent, at least about 80 percent, at least about 80 percent, at least about 80 percent, Comprising a sequence that is 95 percent, at least about 96 percent, at least about 97 percent, at least about 98 percent, at least about 99 percent, or 100 percent nucleotide sequence identity, wherein the sequence is optionally any of the first three bases Including one or more 2′-O-methyl or 2′-F modifications, NA may be further modified as desired. In some embodiments of the Cas9 ribonucleoprotein, the tracrRNA comprises at least about 75 percent, at least about 80 percent, at least about 80 percent, at least about 80 percent, at least about 80 percent, at least about 80 percent, at least about 80 percent, at least about 80 percent, Comprising a sequence that is 95 percent, at least about 96 percent, at least about 97 percent, at least about 98 percent, at least about 99 percent, or 100 percent nucleotide sequence identity, the sequence optionally comprising 2 in the first 3 bases Comprising a modification of '-O-methyl or 2'-F, the tracrRNA optionally It may be modified in La.

In some embodiments of the Cas9 ribonucleoprotein, the crRNA is
PCSK9 crRNA sequence 1:
GGUGCUAGCCUUGCGUUCCGGUUUUAAGAGUAUGCUG (SEQ ID NO: 885), optionally including a modification of 2'-O-methyl or 2'-F in any one or more of the last three bases (GCU),
PCSK9 crRNA sequence 2:
CGUGCUCGGGUGCUUCGGCCGUUUUGAGCUUAUGCUG (SEQ ID NO: 886), optionally including a modification of 2'-O-methyl or 2'-F in any one or more of the last three bases (GCU),
PCSK9 crRNA sequence 3:
GCCGUCCUCCUCGGAACCGCAGUUUUAAGAGCUAUUGCUG (SEQ ID NO: 887), optionally including modification of 2′-O-methyl or 2′-F in any one or more of the first 3 of the last 4 bases (GCU),
PCSK9 crRNA sequence 4:
GGACGAGGACGGCGACUACGGGUUUUAAGAGCUAUUGCUG (SEQ ID NO: 888), optionally including modification of 2'-O-methyl or 2'-F in any one or more of the last three bases (GCU),
PCSK9 crRNA sequence 5:
ACACCCGGGGAAAUCGAGGGCGUUUUAAGAGCUAUUGCUG (SEQ ID NO: 889), optionally including modification of 2′-O-methyl or 2′-F in any one or more of the last three bases (GCU),
PCSK9 crRNA sequence 6:
CGACUUCGAGAAUGUGCCCCGUUUUAAGAGCUAUUGCUG (SEQ ID NO: 890), optionally including modification of 2'-O-methyl or 2'-F in any one or more of the last three bases (GCU),
PCSK9 crRNA sequence 7:
GAGUGACCACCGGGAAAUCGGGUUUUAAGAGCUAUUGCUG (SEQ ID NO: 891), optionally including modification of 2′-O-methyl or 2′-F in any one or more of the last three bases (GCU),
PCSK9 crRNA sequence 8:
CUCGGGCACAUUCUCGAAGUGUUUAGAGCUAUUGCUG (SEQ ID NO: 892), optionally including 2′-O-methyl or 2′-F modifications in any one or more of the last three bases (GCU),
PCSK9 crRNA sequence 9:
GGAAGCCAGGAAGAAGGCCAGUUUAGAGCUAUUGCUG (SEQ ID NO: 893), optionally including 2'-O-methyl or 2'-F modifications in any one or more of the first three of the last four bases (GCU),
PCSK9 crRNA sequence 10:
UCUUUGCCCCAGAGUCUCCCGGUUUUAAGAGCUAUUGCUG (SEQ ID NO: 894), optionally including modifications of 2'-O-methyl or 2'-F in any one or more of the last three bases (GCU), and PCSK9 crRNA sequence 11 :
CUAGGAGAUAACACCUCCCACCGUUUUAGAGCUAUUGCUG (SEQ ID NO: 895), optionally including any modification of 2'-O-methyl or 2'-F in any one or more of the last three bases (GCU), The crRNA is at least about 75 percent, at least about 80 percent, at least about 85 percent, at least about 90 percent, at least about 95 percent, at least about a sequence selected from those that may optionally be further chemically modified. It includes sequences that are about 96 percent, at least about 97 percent, at least about 98 percent, at least about 99 percent, or 100 percent nucleotide sequence identity.

In some embodiments of the Cas9 ribonucleoprotein, the crRNA is
PCSK9 crRNA sequence 1:
GGUGCUAGCCUUGCGUUCCGGUUUUAGAGCUAUUGCUG (SEQ ID NO: 885), optionally including a 2'-O-methyl or 2'-F modification in the first 3 of the last 4 bases (GCU),
PCSK9 crRNA sequence 2:
CGUGCUCGGGUGCUUCGGCCGUUUUAGAGCUAUUGCUG (SEQ ID NO: 886), optionally including a 2'-O-methyl or 2'-F modification in the first 3 of the last 4 bases (GCU);
PCSK9 crRNA sequence 3:
GCCGUCCUCCCUGGAACCGCAGUUUUAAGAGCUAUUGCUG (SEQ ID NO: 887), optionally including 2′-O-methyl or 2′-F modifications in the first 3 of the last 4 bases (GCU),
PCSK9 crRNA sequence 4:
GGACGAGGACGGCGACUACGGGUUUUAAGAGCUAUUGCUG (SEQ ID NO: 888), optionally including a 2'-O-methyl or 2'-F modification in the first 3 of the last 4 bases (GCU),
PCSK9 crRNA sequence 5:
ACACCCGGGGAAAUCGAGGGCGUUUUAAGAGCUAUUGCUG (SEQ ID NO: 889), optionally including 2′-O-methyl or 2′-F modifications in the first three of the last 4 bases (GCU),
PCSK9 crRNA sequence 6:
CGACUUCGAGAAUGUGCCCCGUUUUAAGAGCUAUUGCUG (SEQ ID NO: 890), optionally including 2'-O-methyl or 2'-F modifications in the first 3 of the last 4 bases (GCU),
PCSK9 crRNA sequence 7:
GAGUGACCACCGGGAAAUCGGGUUUUAAGAGCUAUUGCUG (SEQ ID NO: 891), optionally including 2′-O-methyl or 2′-F modifications in the first three of the last 4 bases (GCU),
PCSK9 crRNA sequence 8:
CUCGGGCACAUCUCUCUGAGUGGUUUUAAGAGCUAUUGCUG (SEQ ID NO: 892), optionally including 2′-O-methyl or 2′-F modifications in the first 3 of the last 4 bases (GCU),
PCSK9 crRNA sequence 9:
GGAAGCCAGGAAGAAGGCCAGUUUUAGAGCUAUUGCUG (SEQ ID NO: 893), optionally including 2′-O-methyl or 2′-F modifications in the first 3 of the last 4 bases (GCU),
PCSK9 crRNA sequence 10:
UCUUUGCCCCAGAGCAUCCCGGUUUUAAGAGCUAUUGCUG (SEQ ID NO: 894), optionally including 2′-O-methyl or 2′-F modifications in the first three of the last four bases (GCU), and PCSK9 crRNA sequence 11:
CUAGGAGAUAACACCUCCCACCGUUUUAGGAGCUAUUGCUG (SEQ ID NO: 895), optionally comprising 2'-O-methyl or 2'-F modifications in the first three of the last 4 bases (GCU), wherein the crRNA optionally further comprises At least about 75 percent, at least about 80 percent, at least about 85 percent, at least about 90 percent, at least about 95 percent, at least about 96 percent, at least about at least about 75% of sequences selected from those that may be chemically modified It includes sequences that are 97 percent, at least about 98 percent, at least about 99 percent, or 100 percent nucleotide sequence identity.

  In some embodiments, the first element of the Cas9 ribonucleoprotein comprises a single guide RNA sequence (sgRNA), and the remainder of the compound leaves the Cas9 ribonucleoprotein with one or more of the sgRNA. Connected by interaction. In some embodiments, the sgRNA comprises 20 or more nucleotides. In some embodiments, the sgRNA comprises at least 8 nucleotides.

  In some embodiments of the Cas9 ribonucleoprotein, the degree of complementarity between the sgRNA and its corresponding target sequence is at least 50%, 60%, 70% when optimally aligned using an appropriate alignment algorithm. 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99 %, Or 100%.

In some embodiments of the Cas9 ribonucleoprotein, the sgRNA is
PCSK9 single guide RNA sequence 1:
GGUGCUAGCCUUGCGUUCCGGUUUAAGAGCUAUUGCUGGAAACAGCAAUAGCAAGUUUAAAAUAGAGCUAGUCCCGUUACACACUUGAAAAAAGUGGCACCGAGUCGGUUGCUUUUUUUUUU
PCSK9 single guide RNA sequence 2:
CUGUGUCGGGGUGCUUCGGCCGUUUAAGAGCUAUUGUGGAAACAGCAUGAGCAAGUUUAAAAUAGGCUAUGCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUUGCUUUUUUUUUUU
PCSK9 single guide RNA sequence 3:
GCCGUCCUCCUCGGAACCGCAGUUUAAGAGCUAUUGCUGGAAACAGCAAUAGCAAGUUUAAAAUAGGGCUAGUCCCGUUAUCAACUUGAAAAAGUGGCACCACCAGUCGGUCUUUUUU (sequence number 898)
PCSK9 guide RNA sequence 4:
GGACGAGGACGGCGACUACGGGUAUUAAGAGCUAUUGCUGGAAACAGCAAUAGCAAGUUUAAAUAAGGGCUAGUCCCGUUAUCAACUUGAAAAAGUGGCACCGAGGUCGUGUCUUUUUU (sequence number 899)
PCSK9 single guide RNA sequence 5:
ACACCCGGGGAAAUCGAGGGCGUAUUAAGAGCUAUUGCUGGAAACAGCAAUAGCAAGUUUAAAUAAGGGCUAGUCCCGUUAUCAACUUGAAAAAGUGGCACCAGAGUCGGGUCUUUUUU (sequence number 900)
PCSK9 single guide RNA sequence 6:
CGACUUCGAGAAUGUGCCCCGGUUUAAGAGCUAUUGCUGGAAACAGCAAUAGCAAGUUUAAAUAAGGGCUAGUCCCGUUAUCAACUUGAAAAAGUGGCACCGAGGUCGUGUCUUUUUU (sequence number 901)
PCSK9 single guide RNA sequence 7:
GAUGACACCACCGGGAAAUCGGGUUUAAGAGCUAUUGCUGGAAACAGCAAUAGCAAGUUUAAAUAAGGGCUAGUCCCGUUAUCAACUUGAAAAAGUGGCACCGAGGUCGUGUCUUUUUU (sequence number 902)
PCSK9 single guide RNA sequence 8:
CUCGGGCACAUUCUCGAAGUGUUUAAGAGCUAUUGCUGGAAACAGCAAUAGCAAGUUUAAAUAAGGGCUAGUCCCGUUAUCAACUUGAAAAAGUGGCACCAGAGUCGUGUCUUUUUU (sequence number 903)
PCSK9 single guide RNA sequence 9:
GGAAGCCAGGAAGAAAGGCCAGUUUAAGAGCUAUUGCUGGAAACAGCAUGAGCAAGUUUAAAAUAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGGUCGUGUUUUUUU (sequence number 904),
PCSK9 single guide RNA sequence 10:
UCUUUGCCCCAGAGCAUCCCGGGUUUAAGAGCUAUUGCUGGAAACAGCAUAGCAAGUUUAAAUAAGGGCUAGUCCCGUUAUCAACUUGAAAAAGUGGCACCAGAGUCGUGUCUUUUUU (sequence number 905)
PCSK9 single guide RNA sequence 11:
CUAGGAGAUAACACCUCCCACCGUUUAAGAGCUAUUGCUGGAAACAGCAAUAGCAAGUUUAAAAUAGGCUAGUCCGUUAUCAACUGUAAAAAUGGCACCAGGAUCGGUGCUUUUUU (sequence number 906)
EMX1 single guide RNA sequence:
GUCACCUCCAAUGACAGUAGGGGUAUUAAGAGCUAUUGCUGGAAACAGCAAUAGCAAGUUUAAAUAAGGGCUAGUCCCGUUAUCAACUUGAAAAAGUGGCACCAGAGUCGUGUCUUUUUU (sequence number 907)
ROSA26 single guide RNA sequence:
CGAACCCUACACAUUCAACGGGUUUAAGAGCUAUUGCUGGAAACAGCAAUAGCAAGUUUAAAAUAGGCUAGUCCGUUAUCAACUGUAAAAAUGUGCACCGAGGUCGUGUCUUUUUUU At least about 95 percent, at least about 96 percent, at least about 97 percent, at least about 98 percent, at least about 99 percent, or 100 percent nucleotide sequence identity.

  In some embodiments of the compounds of the invention, Y is a Cpf1 ribonucleoprotein. In some embodiments, Y is (1) a first element that includes a recognition element that includes a guide sequence, which upon expression expresses sequence-specific binding of Cpf1 ribonucleoprotein to a target sequence. The first element being directed, and (2) a Cpf1 ribonucleoprotein comprising a second element comprising a Cpf1 protein and optionally one or more nuclear localization sequences (NLS), wherein the first element Is associated with the second element. In some embodiments, the target sequence is a eukaryotic target sequence. In some embodiments, the target sequence is a prokaryotic target sequence.

In some embodiments, the Cpf1 ribonucleoprotein comprises crRNA, wherein the crRNA is
EMX1 crRNA sequence (Asp Cpf1):
UAAUUUCUACUCUGUUGAUUCAUCUUGUGCCCCUCCCUCCCUG (SEQ ID NO: 909),
EMX1 crRNA sequence (Lba Cpf1):
UAAUUUCUACUCUUAAGUAGAUUCAUCUUGUGCCCCUCCCUCCCUG (SEQ ID NO: 910),
PCSK9 crRNA sequence 12 (Asp Cpf1):
UAAUUUCUACUCUGUGUAUCCCAGAGCAUCCCGUGGACACCUG (SEQ ID NO: 911),
PCSK9 crRNA sequence 12 (Lba Cpf1):
UAAUUUCUACUCUAUAGUAGUCCCAGAGCAUCCCGUGGACACCUG (SEQ ID NO: 912),
PCSK9 crRNA sequence 13 (Asp Cpf1):
UAAUUUCUACUCUGUGUAGUCCCGUGGUCACUCUGUAUUGCUGG (SEQ ID NO: 913), and PCSK9 crRNA sequence 13 (Lba Cpf1):
A sequence selected from UAAUUUCCUCUUAAGUAGUCCCGUGUGUCACUCUGUAUUGCUGG (SEQ ID NO: 914), which is optionally chemically modified, at least about 75 percent, at least about 80 percent, at least about 85 percent, at least about 90 percent, at least about It includes sequences that are 95 percent, at least about 96 percent, at least about 97 percent, at least about 98 percent, at least about 99 percent, or 100 percent nucleotide sequence identity.

  In some embodiments of the compounds described herein, Y is a type III-B Cmr complex, eg, a type III-B Cmr complex from Pyrococcus furiosus, Sulfolobus solfarricus, and Thermus thermophilus. In some embodiments, Cmr proteins suitable for use herein include Hale, C .; R. et al. Genes & Development, 2014, 28: 2432-2443, and Makarova K. et al. S. et al. Examples include, but are not limited to, those described in Nature Reviews Microbiology, 2015, 13, 1-15.

  In some embodiments of the compounds described herein, Y further comprises a fluorescent probe. In some embodiments, the fluorescent probe comprises an mCherry sequence (SEQ ID NO: 915). Other suitable fluorescent proteins include green fluorescent protein (GFP) or variants thereof, GFP blue fluorescent variant (BFP), GFP cyan fluorescent variant (CFP), GFP yellow fluorescent variant (YFP), High-sensitivity GFP (EGFP), high-sensitivity CFP (ECFP), high-sensitivity YFP (EYFP), GFPS65T, emerald, topaz (TYFP), Venus, Citriline, mCitrine, GFPuv, destabilized EGFP (dEGFP), destabilized ECFP (DECFP), destabilized EYFP (dEYFP), mCFPm, Cerulean, T-Sapphire, CyPet, YPet, mKO, HcRed, t-HcRed, DsRed, DsRed2, DsRed-monomer, J-Red, dimer 2, t-dimer 2 12), phycobiliproteins and phycobiliprotein conjugates such as mRFPl, pocilporin, Renilla GFP, Monster GFP, paGFP, Kaede protein and kindling protein, B-phycoerythrin, R-phycoerythrin, and allophycocyanin. It is not limited to these. Other examples of fluorescent proteins include mHoneydew, mBanana, mOrange, dTomato, tdTomato, mTangerine, mStrawberry, mCherry, mGrape1, mRaspberry, mGrape2, mPrape, mGrape, S Is mentioned. For example, Matz et al. (1999) Nature Biotechnol. 17: 969-973, any of a variety of fluorescent and colored proteins derived from an insect species is suitable for use.

  In some embodiments, Y includes one or more NLS. In some embodiments, Y comprises one or more NLS, each independently an NLS of the SV40 viral large T antigen having the amino acid sequence PKKKRKV (SEQ ID NO: 830), an NLS from nucleoplasmin (eg, Nucleoplasmin bisection NLS of the sequence KRPAATKKAGQAKKKKK (SEQ ID NO: 831), c-myc NLSN having the amino acid sequence PAAKRVKLD (SEQ ID NO: 832) or RQRRNELKRSP (SEQ ID NO: 833), the sequence NQSSNNGPMKGNGGGSGGPGG The sequence RMRIXFKNKKGKDTAELRR is any amino acid (SEQ ID NO: 835) of the IBB domain from importin alpha RVESVVERKAKKKDEQILKRNV, myoma T protein sequences VSRKRPRP (SEQ ID NO: 836) and PPKKARED (SEQ ID NO: 837), human p53 sequence PQPKKKKPL (SEQ ID NO: 838), mouse c-abl IV sequence SALIKKKKKKMAP (SEQ ID NO: 839), influenza N1 Sequences DRLRR (SEQ ID NO: 840) and PKQKKRK (SEQ ID NO: 841), hepatitis delta virus antigen sequence RKLKKKIKKL (SEQ ID NO: 842), mouse Mx1 protein sequence REKKKKLKRR (SEQ ID NO: 843), human poly (ADP-ribose) polymerase sequence KRKGDEVDGVDEVAKKKSKK (SEQ ID NO: 844), steroid hormone receptor (human) glucocorticoid An embodiment comprising, but not limited to, NLS including, but not limited to, NLS sequences derived from the sequence RKCLQAGMNLEARKTKK (SEQ ID NO: 845), the sequence MAPKKKKRKVGIHRGVP (SEQ ID NO: 1035), and the sequence PKKKRKVEDPKKKKRKVD (SEQ ID NO: 1036). There may optionally be an intervening amino acid sequence between each of the two NLSs, such intervening sequences may include one or more amino acid residues, in some embodiments, Y is two Contains NLS, each containing the amino acid sequence PKKRKVV (SEQ ID NO: 830) Y contains 3 NLS, each containing the amino acid sequence PKKRKV (SEQ ID NO: 830).

  In some embodiments of the compounds of the invention, Y represents S. aureus, S .; pneumoniae, S .; pyogenes, S .; thermophilus, S. et al. Aureus, N.M. meningitidis, or A. at least about 75 percent, at least about 80 percent, at least about 85 percent, at least about 90 percent, at least about 95 percent, at least about 96 percent, at least about 97 percent, at least about 98 percent, at least about Cas9 protein from ebreus It contains a Cas9 protein that is about 99 percent, or 100 percent amino acid sequence identity. See, for example, Hou et al, PNAS 2013. In some embodiments, Y is at least about 75 percent, at least about 80 percent, at least about 85 percent, at least about 90 percent, at least about 95 percent, at least about 96 percent, at least about Type II Cas9 protein. A Cas9 protein that is about 97 percent, at least about 98 percent, at least about 99 percent, or 100 percent amino acid sequence identity. In some embodiments, Y is at least about 75 percent relative to amino acids at positions 7-166, or 731-1003 of SEQ ID NO: 8, or the corresponding amino acids of those set forth in SEQ ID NOs: 1-7, 9-829. At least about 80 percent, at least about 85 percent, at least about 90 percent, at least about 95 percent, at least about 96 percent, at least about 97 percent, at least about 98 percent, at least about 99 percent, or 100 percent amino acid sequence identity Contains certain Cas9 proteins. In some embodiments, Y is at least about 75 percent, at least about 80 percent, at least about 85 percent, at least about 90 percent, at least about amino acids at positions 7-166, or 731-1003 of SEQ ID NO: 8. A Cas9 protein that is about 95 percent, at least about 96 percent, at least about 97 percent, at least about 98 percent, at least about 99 percent, or 100 percent amino acid sequence identity. In some embodiments, Y comprises a Cas9 protein having at least four motifs in the sequence, the motif comprising motifs 1, 2, 3, and any of the Cas9 amino acid sequences of any of SEQ ID NOs: 260-263, and 4, at least about 75 percent, at least about 80 percent, at least about 85 percent, at least about 90 percent, at least about 95 percent, at least about 96 percent, at least about 97 percent, at least about 98 percent, at least about 99 percent, Or 100 percent amino acid sequence identity.

In some embodiments, Y comprises a Type II-C Cas9 protein. In some embodiments, Y is S.I. pyogenes Cas9 (wild type) (SEQ ID NO: 848), S. pyogenes Cas9 mutant M1C (SEQ ID NO: 849), S. cerevisiae. pyogenes Cas9 mutants M1C and C80S (SEQ ID NO: 850), S. cerevisiae. pyogenes Cas9 nickase mutant D10A (SEQ ID NO: 851), S. cerevisiae. pyogenes Cas9 nickase mutant H840A (SEQ ID NO: 852), S. pyogenes Cas9 nickase variants E923P and T924P (SEQ ID NO: 853), Acidovorax ebreus Cas9 (SEQ ID NO: 854), acidic mine drainage bacterium Ga0052161_JGI Cas9 (SEQ ID NO: 855), S. at least about 75 percent, at least about 80 percent, at least about 85 percent, at least about a sequence selected from pyogenes Cas9 null mutants D10A and H840A (SEQ ID NO: 1027), and the uranium mine bacterium FW106_JGI Cas9 (SEQ ID NO: 856) A Cas9 protein that is about 90 percent, at least about 95 percent, at least about 96 percent, at least about 97 percent, at least about 98 percent, at least about 99 percent, or 100 percent amino acid sequence identity.

  In some embodiments of the Cas9 protein suitable for use herein, the Cas9 protein sequence includes modifications to the sequence for expression in a eukaryotic cell or to affect its function. Thus, for example, it can be modified as codon optimization. In some embodiments, the Cas9 protein sequence targets cleavage of one or two DNA strands, eg, guide sequence (s), eg, the sense and antisense strands of the DNA target, respectively. This causes both strands to be cleaved and aligned to that position of the target, such as with Cas9 nickase (ie, Cas9-D10A) used in combination with two guide sequences that result in non-homologous end joining. Cas9-D10A and single guide RNA sequences can create indels. However, in other embodiments, the Cas9 protein sequence lacks DNA strand cleavage activity, such as by selective use of a non-catalytic Cas (dCas) domain. In other embodiments, the Cas9 protein sequence is modified, including lysine, glutamine, and cysteine residue modifications, to confer a covalent bond to the Cas9 protein or Cas9 ribonucleoprotein. In yet other embodiments, the Cas9 ribonucleoprotein is O'Connell, Mitchell R. et al. , Et al. , Programmable RNA recognition and cleavage by CRISPR / Cas9, Nature, 2014, 516, p263-266, can be used to direct RNA strand cleavage.

  In another embodiment, the Cas9 protein comprises a mutated residue, eg, a solvent-exposed lysine or arginine residue mutated to histidine, cysteine, or glutamine (not involved in catalytic activity, DNA binding, or RNA binding). Have.

  Modification of the Cas9 protein sequence can include a mutation of D10A (aspartic acid to alanine at amino acid position 10 of SEQ ID NO: 8) (or any corresponding mutation of the protein shown as sequences 1-829), This can cleave the complementary strand of the target DNA, but has a reduced ability to cleave the non-complementary strand of the target DNA (thus resulting in single strand breaks instead of double strand breaks). Another modification is a mutation of H840A (histidine to alanine at amino acid position 840 of SEQ ID NO: 8) (or the corresponding mutation of any of the proteins shown as sequences 1-829), which is non-complementary to the target DNA. Although the strand can be cleaved, the ability to cleave the complementary strand of the target DNA is reduced (thus resulting in single strand breaks instead of double strand breaks). In contrast to single-strand breaks, the presence of double-strand breaks greatly increases the likelihood of non-homologous end joining, so either the D9A or H840A variant of Cas9 (or any of the proteins shown as sequences 1-829) The use of corresponding mutations in can change the expected biological effect.

  Other residues can be mutated to inactivate specific nucleases from motif 1, 2, 3, or 4 as well. As non-limiting examples, the correspondence in residues D10, G12, G17, E762, H840, N854, N863, H982, H983, A984, D986, and / or A987 (or any of the proteins shown as SEQ ID NOs: 1-829) Mutations) can be modified. Mutations can include substitutions, additions and deletions, or any combination thereof. In some examples, the mutation converts the mutant amino acid to another amino acid, such as alanine. Other modifications include base modifications, backbone modifications, and / or internucleoside linkage modifications.

  In some embodiments of the compounds of the invention, Y is Y1C80S-3N-m or Y1C80S-2N. In some embodiments, Y is Y1C80S-3N-m-RNP-EMX1. In some embodiments, the compound is Y53aASGPRL. In some embodiments, the compound is Y53aASGPRL-RNP-EMX1, Y53aASGPRL-RNP-PCS4, or Y53aASGPRL-RNP-PCSl.

  In some embodiments of the compounds of the invention, Y represents A.I. sp. , L. bacterium, p. macacae, and P.I. at least about 75 percent, at least about 80 percent, at least about 85 percent, at least about 90 percent, at least about 95 percent, at least about 96 percent, at least about 97 percent, at least about 98 percent, at least about Cpf1 protein from disiens Includes Cpf1 proteins that are about 99 percent, or 100 percent amino acid sequence identity. In some embodiments, Y is A.I. sp Cpf1 (SEQ ID NO: 857), L. Bacterium Cpf1 (SEQ ID NO: 858), P.I. macacae Cpf1 (SEQ ID NO: 859), P. a. at least about 75 percent, at least about 80 percent, at least about 85 percent, at least about 90 percent, at least about 95 percent, at least about 96 relative to a Cpf1 protein selected from but not limited to disiens Cpf1 (SEQ ID NO: 860) A Cpf1 protein that is percent, at least about 97 percent, at least about 98 percent, at least about 99 percent, or 100 percent amino acid sequence identity.

  In some embodiments, the compounds of the invention further comprise an endosomal escape agent. In some embodiments of the compounds of the invention, the Y portion of the compound further comprises an endosomal escape agent. In some embodiments, the endosomal escape agent is linked to the remainder of the compound via one or more covalent bonds. In some endosomal embodiments, the endosomal escape agent is associated with the remainder of the compound, for example, via electrostatic interactions. In some embodiments, the endosomal escape agent is co-incubated with a compound of the invention.

  Endosome escape agents suitable for use in the present invention include, but are not limited to, lysosome agonists, cell permeable peptides, membrane fusion peptides, endosome lytic peptides, pore formers, and proton sponge agents.

The lysosome agonist is a weak base and can penetrate the lysosome as a protonated form, raising the intracellular pH. Suitable lysosomal agents for use in the present application include chlorpromazine, amantadine, 4-aminoquinoline, amiodarone, amodiaquine, azithromycin, chloroquine, clindamycin, N- (3-[(2,4-dinitrophenyl)- Amino] -propyl) -N- (3-aminopropyl-methylamine) dihydrochloride (DAMP), imipramine, methylamine, monensin, monodansyl cadaverine, NH ₄ Cl, perhexylene, phenylalanine methyl ester, primaquine, quinacrine, suramin , Thioridazine, tyrolone, tributylamine, ketotifen fumarate, glycerol, and sucrose. In some embodiments, the lysosomal agent is chloroquine, glycerol, or sucrose.

Cell penetrating peptides (CPPs) are a diverse class of peptides, usually comprising 5 to 30 amino acids that can penetrate the cell membrane. In some embodiments, CPPs suitable for use herein include cationic CPPs, amphiphilic CPPs, and hydrophobic CPPs. CPPs suitable for use herein include CPPs derived from heparin binding, RNA binding, and DNA binding proteins such as those shown in Table 1, signal peptide derived CPPs such as those shown in Table 2, and Table 3 CPPs derived from antimicrobial peptides such as those shown, CPPs derived from viral proteins such as those shown in Table 4, CPPs derived from various natural proteins such as those shown in Table 5, and designed CPPs such as those shown in Table 6 And CPPs derived from peptide libraries. For example, Milletti, F.M. See Drug Discovery Today, Volume 17, Numbers 15/16, 2012, 850-860.

  Membrane fusion peptides are short peptides that destabilize phospholipid membranes. In some embodiments, the membrane fusion peptide comprises 20-30 amino acids. They trigger endosomal escape mainly by two main mechanisms: membrane fusion and pore formation. Membrane fusion peptides suitable for use herein include influenza HA-2 peptide, GLFGAIAGFIENGWEGMIDGWYG (SEQ ID NO: 863), melittin, GIGAVLKVLTGLLPALISWIKRKRQQ (SEQ ID NO: 864), tat (48-60), GRKKRRQRRPQ (SEQ ID NO: RQRRPQ) Penetratin, RQKIKIWFQNRRMKWKK (SEQ ID NO: 866), Transportan, GWTLNSAGYLLGKINLKALAALALAKKIL (SEQ ID NO: 867), GALA peptide, WEALAEALAEALALAHLAEALAEALEALAA (SEQ ID NO: 868), KALAALALALALALA LELLESLWELLLEA (SEQ ID NO: 870), ppTG1, GLFKALLKLLKSLWKLLLKA (SEQ ID NO: 871), ppTG20, GLFRALLRLLRSLWRLLLRA (SEQ ID NO: 872), and derivatives thereof are not limited thereto. In some embodiments, the endosomal escape agent comprises the KALA peptide, WEAKLAKALAKALAKHLAKALAKALKACEA (SEQ ID NO: 869).

In some embodiments, the endosomal escape agent is an endosome selected from, but not limited to, CHK ₆ HC (SEQ ID NO: 861), H5WYG (SEQ ID NO: 1029), GLFHAIAHFIHGGWHGLIHGGWYG (SEQ ID NO: 862), and derivatives thereof. Contains dissolved histidine-rich peptide. In some embodiments, the endosomal escape agent comprises a peptide of H5WYG (SEQ ID NO: 1029), or GLFHAIAHFIHGGWHGLIHGGWYG (SEQ ID NO: 862).

In some embodiments, the endosomal escape agent is a synthetic peptide. In some embodiments, the endosomal escape agent has the structure:
The peptide ppTG21: GLFHALLHLLHSLWHLLLLHA (SEQ ID NO: 1012) has a hydrogen atom omitted from the peptide structure for clarity. Rittner, Karola, et al. , New Basic Membrane-Destabilizing Peptides for Plasmid-Based Gene Delivery in Vitro and in Vivo, MOLECULAR THERAPY 5 (2) 104-114 (2002).

  A pore-forming agent is an agent such as a peptide that induces pore formation through the membrane and thereby destroys the endosome. Suitable pore-forming agents for use herein include cecropin (insect), magainin, CPF 1, PGLa, bonbinine BLP-1 (all three from amphibians), melittin (bee), seminal plasmin ( Cattle), indolicidin, bactenecin (both from bovine neutrophils), tachypressin 1 (crab), protegrin (pig leukocytes), and defensin (from humans, rabbits, cows, fungi, and plants), gramicidin A and gramicidin S ( Bacillus brevis), lantibiotics such as, but not limited to, nisin (lactococcus lactis), androctonine (scorpion), cardiotoxin I (cobra), frog lintoria splendida, dermaseptin (frog). Viral peptides have also been shown to have pore-forming activity, such as hemagglutinin subunits HA-2 (influenza virus), E1 (Semliki Forest virus), F1 (Sendai and measles virus), gp41 (HIV), gp32 (SIV), and vp1 (rhino, polio, and coxsackie viruses). In some embodiments, the endosomal escape agent is a rhinovirus-derived vp1 peptide. In some embodiments, the endosomal escape agent is a bee venom-derived melittin peptide, or a masked analog thereof.

  A proton sponge agent is a drug having a plurality of proton receptor sites that destroy endosomes by osmolytic action. In some embodiments, a proton sponge agent suitable for use herein comprises a plurality of proton receptor sites having a pKa value between physiological pH and lysosomal pH. In some embodiments, a proton sponge agent suitable for use herein comprises a plurality of proton acceptor sites having a pKa value in the range of 4-7, wherein the endosomal lytic component is a polycation at pH 4 It is sex. In some embodiments, the endosomal escape agent is a proton sponge agent selected from imidazole-containing compounds such as compounds or peptides comprising one or more histidines, histamines, vinyl imidazoles, or combinations thereof. In some embodiments, proton sponge agents suitable for use herein have one or more secondary or tertiary amines and have a pKa value between physiological pH and lysosomal pH. The polymer which shows is mentioned. In some embodiments, proton sponge agents suitable for use herein include polyethyleneimine, polyamidoamine dendrimers, PEG-oligo (glutamic acid) -PEI, poly (L-histidine), chloroquine, methylamine, A polymer such as ammonium chloride may be mentioned, but is not limited thereto.

  In another embodiment, the described compounds are capable of binding to receptors present on hepatocytes. In another embodiment, the receptor present on hepatocytes is an asialoglycoprotein receptor.

Methods of Preparation The compounds of the present invention can be synthesized by synthetic routes involving processes similar to those well known in the chemical art, especially in light of the description contained herein. Starting materials are generally available from commercial sources such as Aldrich Chemicals (Milwaukee, Wis.), Or are readily prepared using methods well known to those skilled in the art (eg, Louis F. Fieser and Mary Fieser, Reagents for Organic Synthesis, v. 1-19, Wiley, New York (1967-1999 ed.), Or Beilsteins Handbuch der organischen Chemie, Ver. (Also available in Stein's online database)).

  For illustrative purposes, the reaction schemes shown below provide potential synthetic routes for the compounds of the present invention and key synthetic intermediates. See the Examples section below for a more detailed explanation of the individual reaction steps. Those skilled in the art will appreciate that other synthetic routes may be used to synthesize the compounds of the invention. Although specific starting materials and reagents are shown in the scheme and discussed below, other starting materials and reagents can be readily substituted to provide various derivatives and / or reaction conditions. In addition, many of the compounds prepared by the methods described below can be further modified in light of this disclosure using conventional chemistry well known to those skilled in the art.

  In preparing the compounds of the present invention, protection of intermediate remote functional groups (eg, primary or secondary amines) may be necessary. The need for such protection depends on the nature of the remote functional group and the conditions of the preparation method. Suitable amino protecting groups (NH-Pg or NPg) include acetyl, trifluoroacetyl, t-butoxycarbonyl (BOC), benzyloxycarbonyl (CBz), 9-fluorenylmethyleneoxycarbonyl (Fmoc), and phthalimide (Pht). “Hydroxy protecting group” refers to a substituent of a hydroxy group that blocks or protects the hydroxy functionality. Suitable hydroxyl protecting groups (O-Pg) include, for example, allyl, acetyl (Ac), silyl (such as trimethylsilyl (TMS) or tert-butyldimethylsilyl (TBS)), benzyl (Bn), para -Methoxybenzyl (PMB), trityl (Tr), para-bromobenzoyl, para-nitrobenzoyl, and the like (benzylidene, cyclic ketal, orthoester to protect 1,2- or 1,3-diol, Orthoamide). The need for such protection is readily determined by one skilled in the art. For an overview of protecting groups and their use, see T.W. W. See Greene, Protective Groups in Organic Synthesis, John Wiley & Sons, New York, 1991.

Scheme 1 outlines a general procedure that can be used to provide compounds of the invention. In Step 1 of Scheme 1, H. Paulsen and M.M. Synthetic intermediate (Ia), which can be prepared by the procedure described in Paal in Carbohydrate Research, 135, 53 (1984), is prepared under classical conditions [room temperature (about 23 ° C.) and trimethylsilyl chloride and pyridine. Persilylation followed by selective cleavage of the trimethylsilyl group protecting the primary alcohol (temperature ranging from about −10 ° C. to room temperature under basic conditions such as potassium carbonate in an alcohol solvent such as methanol). The primary alcohol intermediate (Ib). In Step 2 of Scheme 1, the additional hydroxymethylene group found in intermediate (Ic) is added to J.I. R. Parikh and William v. E. Doering in Journal of the American Chemical Society, 89, 5505-5507 (1967), followed by Parikh-Doering oxidation in water or alcohol solvents at temperatures ranging from about room temperature to about 60 ° C. This glycoside can be introduced by reaction with a formaldehyde source (eg, aqueous formaldehyde solution, solid paraformaldehyde) in the presence of an oxide (eg, sodium hydroxide, sodium alkoxide). This is called the Aldol-Canizzaro reaction. Modifications of this process known to those skilled in the art may be used as well. For example, other oxidizing agents such as Ozanne, A. et al. et al. in Organic Letters, 5, 2903 (2003), stabilized 2-iodoxybenzoic acid, Kanji Omura and Daniel Swern in Tetrahedron, 34, 1651 (1978), and others known to those skilled in the art The oxidizing agent can also be used in the same manner. The Aldol Canizzaro sequence is described by Robert Schaffer in the Journal of The American Chemical Society, 81, 5452 (1959), and Amiges, E. et al. J. et al. In Tetrahedron, 63, 10042 (2007). The experimental conditions of Step 2 of Scheme 1 also facilitate the cleavage of the trimethylsilyl group protecting the secondary alcohol. In step 3, scheme 1, intermediate (Ic) is reacted with an organic or inorganic acid (eg, sulfuric acid) or acidic resin in a solvent such as water at a temperature ranging from about room temperature to about 100 ° C. (1) is obtained. In step 4 of Scheme 1, compound (1) can be reacted with a reducing agent known to reduce the azide group to the corresponding amine (eg, transition metal mediated catalytic hydrogenation, a classic well known to those skilled in the art). Using triphenylphosphine in water under experimental conditions). Compound (2) is then reacted in the presence of an acylating agent (eg, acetic anhydride or acetyl chloride in a solvent such as dichloromethane or tetrahydrofuran in the presence of pyridine or triethylamine at temperatures ranging from 0 to 80 ° C.). obtain.
In step 5 of Scheme 1, compound (2) is reacted with compound (2) in the presence of an alkoxide (eg, sodium methoxide) in a solvent or solvent mixture such as an alcohol solvent or tetrahydrofuran at a temperature ranging from about 0 to room temperature. 3) is obtained. Furthermore, the compounds thus obtained can then be readily functionalized to compounds according to other claims of the invention using well-known protection and functional group manipulation sequences known to those skilled in the art. it can. Accordingly, in steps 6 and 7 of Scheme 1, the secondary hydroxyl groups of compounds (1) and (3), respectively, are further protected with a suitable protecting group (eg, N, at temperatures ranging from about room temperature to about 90 ° C.). Intermediates such as (Id) and (Ie) can be obtained as a cyclic ketal by reaction with 2,2-dimethoxypropane in a solvent such as N-dimethylformamide under acidic conditions. Next, using synthetic transformations and manipulations of functional groups and protecting groups well known to those skilled in the art, (Id) and (Ie) are prepared for further functionalization and derivatization of the primary hydroxyl group, The desired linker X and ligand Y of interest are linked to produce an XY-containing compound according to the claims of the present invention. Removal of protecting groups (eg, Pg) using reagents and conditions well known in the art to reveal secondary hydroxyl groups (eg, when two Pg forms a cyclic ketal such as acetonide, acetic acid, alcohol solvent And can be removed under acidic conditions using an acid such as acetic acid in a solvent or solvent mixture such as water, tetrahydrofuran, etc., at temperatures ranging from room temperature to about 80 ° C.). It leads to XY-containing compounds. For example, after manipulation and removal of the protecting groups, alkylation of the primary hydroxyl group of (Ie) can result in the corresponding ether-linked XY-containing compound according to the claims of the present invention. Similarly, the XY-containing compounds of the ester bond, carbonate bond, and carbamate bond according to the claims of the present invention can be obtained from (3) or intermediate (Ie) using appropriate reactants and reagents well known to those skilled in the art. And can be obtained easily. After manipulation and removal of the protecting groups by conversion of the primary hydroxyl group of (Ie) to the corresponding triflate (III-e-1) followed by nucleophilic substitution with a suitable nucleophile, the present invention The corresponding ether and thioether linked XY-containing compounds according to the claims can be provided. The oxidation of the thioether intermediate can also result in the corresponding sulfoxide and sulfone linked XY-containing compounds according to the claims of the present invention. Furthermore, substitution of the primary triflate of (III-e-1) with potassium thioacetate followed by hydrolysis of the thioester can give the corresponding thiol (III-e-2), which is a protecting group. After manipulation and removal of thioether-linked XY according to the claims of the present invention, the compound (IV-e-1) is obtained and further alkylation of the thiol (III-e-2) and manipulation and removal of the protecting groups. Containing compounds can be produced. (III-e-2) can also be converted to the corresponding sulfonyl chloride and reacted with the appropriate amine to produce the sulfonamide-linked XY-containing compounds of the present invention after manipulation and removal of the protecting groups. This primary triflate (III-e-1) can also be replaced with sodium azide to produce the corresponding azide-containing compound (III-e-3), which involves manipulation and removal of protecting groups. To give compound (IV-e-2). Reduction of compound (III-e-3), after manipulation and removal of protecting groups, XY substituents are linked to further functionalize (eg, amide bond formation) to produce compounds according to the claims of the present invention. The corresponding primary amines (III-e-4) for reductive amination, sulfonamide formation, urea formation, carbamate formation, etc.). (III-e-4) can also produce the compound (IV-e-3) of the present invention after manipulation and removal of the protecting group. Reaction of the above azide intermediate (III-e-3) with an alkyne or nitrile-containing reagent or synthetic intermediate, followed by manipulation and removal of the protecting group under conditions well known to those skilled in the art, respectively. An XY-containing compound having a triazole or tetrazole bond according to the claims can be produced.
Oxidation of the primary hydroxyl group of (Ie) to the corresponding aldehyde (III-ed) followed by reductive amination with an appropriate amine under classical conditions known to those skilled in the art, or Olefination (eg, Wittig, Horner Wadsworth Emmons, Peterson, Julia type and modifications thereof), followed by reduction of the formed olefin (eg, metal mediated catalytic hydrogenation or diimide well known to those skilled in the art) With the aid of intervening reduction, after the manipulation of the functional group and the manipulation and removal of the protecting group, respectively, the desired nitrogen or carbon-bonded XY-containing compounds according to the claims of the invention can be brought about. Conversion of this aldehyde (III-e-5) to the corresponding alkyne (III-e-6) (using a Cory-Fuchs type reaction or using a Seyfers-Gilbert type reagent) followed by a protecting group And removal can yield compound (IV-e-4). The reaction of the alkyne (III-e-6) or (IV-e-4) with an azide-containing reagent or synthetic intermediate followed by manipulation and removal of the protecting group under conditions well known to those skilled in the art. Similarly, triazole-linked XY-containing compounds according to the claims of the present invention can be produced. Alkynes (III-e-6) also function as useful synthetic intermediates, which can be reacted with appropriate reagents under metal-mediated cross coupling (eg, Sonogashira reaction) known to those skilled in the art. Other compounds according to the claims of the invention can be obtained. Oxidation of the primary hydroxyl group of (Ie) to the corresponding acid (III-e-7) using synthetic transformations well known to those skilled in the art is subject to the claims of this invention after removal of the protecting group. Provides access to XY-containing compounds with ester and amide linkages. Manipulation and removal of the protecting group of (III-e-7) also provides easy access to compound (IV-e-5). Conversion of (IV-e-5) or (III-e-7) to the corresponding primary amide under conditions well known to those skilled in the art can be accomplished directly from compound (IV-e-6) or manipulation of protecting groups. And compound (III-e-8) which gives (IV-e-6) after removal. Furthermore, dehydration of the amide functional group of (III-e-8) can give the corresponding nitrile (III-e-9), which, after manipulation and removal of the functional group, is compound (IV-e -7). Substitution of the primary triflate (III-e-1) with a cyanide anion can similarly produce the corresponding nitrile-containing compound (III-e-10), which is after manipulation and removal of the protecting groups. Gives compound (IV-e-8). Next, hydrolysis of the nitrile of (IV-e-8) or (III-e-10) directly to the acid (IV-e-9) or after manipulation and removal of the protecting group (IV -E-9) can be obtained to obtain (III-e-11).
Alkyne-containing compounds such as (III-e-6) / (IV-e-4), primary amide-containing compounds such as (III-e-8) / (IV-e-6), (III-e-9) Nitrile-containing compounds such as / (IV-e-7) / (III-e-10) / (IV-e-8), (III-e-7) / (IV-e-5) / (III-e -11) / (IV-e-9) and other acid-containing compounds, and (III-e-5) and other aldehyde-containing compounds are also functionalized to provide appropriate reagents and synthetic intermediates well known to those skilled in the art ( and, J.A.Joule and K.Mills, Heterocyclic Chemistry, 5 th edition, Wiley Ed., (2010), J.J.Li, Name Reactions in heterocyclic chemistry, Wil y, (2005), MR Grimmett Advances in Heterocyclic Chemistry, 27, 241, (1981), IG Turchi et al., Chemical Reviews, 75, 389, (1975), T.T. Chemical Reviews, 61, 87, (1961), R. H. Wiley, Organic Reactions, 6, 367, (1951), L. B. Clap, Advances in Heterocyclic Chemistry, 20, 65, H, h, 76, h, 76A. et al., Advances in Heterocyclic Chemistry, 7, 183, (1967), J. Sandstrom, Advanced ce in Heterocyclic Chemistry, 9, 165, (1968), S. J. Wittenberger, Organic Preparations and Procedures International, 26, 499, (1994), M. G. Finn et al. , (Summarized in (2009)) and further 5- and 6-membered rings according to the claims of the present invention (eg isoxazole, isothiazole, pyrazole, oxazole, thiazole, imidazole, 1,2, , 4-oxadiazole, 1,2,4-thiadiazole, 1,2,4-triazole, 1,3,4-oxadiazole, 1, , 4-thiadiazole, tetrazole, 1,2,3-triazole) can be prepared XY-containing compound of the bond. XY-containing compounds with aryl ring bonds according to the claims of the present invention are likewise from alkynes such as (III-e-6) and (IV-e-4) or hetero-substituted analogues of these alkynes (ie By substituting the alkyne hydrogen of (III-e-6) / (IV-e-4) with OR ⁴ , N (R ⁴ ) ₂ , SR ^4. These compounds are prepared under conditions and reagents known to those skilled in the art. Can be obtained via a benzannulation reaction known to those skilled in the art (for example, Danheiser-type or Detz-type benzannulation).

  Similar chemistry as described above for compounds (3) and (Ie) can be applied to compound (1) and intermediate (Id) as well to give further compounds according to the claims of the present invention. . See the Examples section for further details.

The use of trifluoroacetic anhydride in Step 4 of Scheme 1 is similarly N-((1S, 2R, 3R, 4R, 5S) -2,3-dihydroxy-1- (hydroxymethyl) -6,8-di Oxabicyclo [3.2.1] octane-4-yl) -2,2,2-trifluoroacetamide, i.e. a method for obtaining compound (24), can be given.

The compounds of the present invention can be further functionalized, reacted, and formulated under conditions known to those skilled in the art, particularly when R ^{1 comprises an} aliphatic, PEG-derived chain, PEG-derived oligomer or polymer. Further compounds according to paragraphs can be obtained and these can be obtained from liver-selective drug delivery systems such as biodegradable PLGA-b-PEG polymer nanoparticles (Erica Locatelli et al., Journal of Nanoparticular Research, 14, 1316, (See 2012)) and lipid-based platforms such as liposomes, lipid nanoparticles, stable nucleic acid lipid nanoparticles (see SaraFalsini et al., Journal of Medicinal Chemistry, 57, 1138 (2014)). There can be incorporated.

  Diastereomeric mixtures are resolved into their individual diastereoisomers on the basis of their physical chemical differences by methods well known to those skilled in the art, for example, by chromatography and / or fractional crystallization, distillation, sublimation. be able to. Enantiomers convert enantiomeric mixtures into diastereomeric mixtures by reaction with appropriate optically active compounds (eg, chiral alcohols, asymmetric auxiliaries such as mosher acid chlorides), and these diastereomers are separated into individual Can be resolved by converting their diastereomers into the corresponding pure enantiomers. Also, some of the compounds of the present invention may be atropisomers (eg, substituted biaryls) and are considered as part of this invention. Enantiomers can also be resolved by use of chiral HPLC (high pressure liquid chromatography) columns.

  The intermediates and compounds of the present invention may exist in different tautomeric forms, and all such forms are included within the scope of the present invention. The term “tautomer” or “tautomer” refers to structural isomers of different energies that are interconvertible with a low energy barrier. For example, proton tautomers (also known as prototrophic tautomers) include interconversions via proton rearrangements, such as keto enol and imine enamine isomerization. A specific example of a proton tautomer is an imidazole moiety, where the proton may rearrange between two ring nitrogens. Valence tautomers include interconversions by reorganization of some of the bonding electrons. The equilibrium between closed and open forms of some intermediates (and / or mixtures of intermediates) is very similar to the process of rotatory rotation involving aldoses known to those skilled in the art.

Scheme 3 illustrates an exemplary Cas9 modification in the preparation of compounds of the invention, where n, o, p, q, or r is an integer independently selected from 0-50 for each occurrence.

Scheme 4 shows an exemplary process for linking via disulfide bonds to Cas9 protein or one or more other parts of Cas9 ribonucleoprotein, where n is independently from 0-50 from each occurrence. An integer that is selected. One skilled in the art will appreciate that the disulfide bonds shown herein can be replaced with other suitable bonds.
“ASGPRL” or “ASGPrL” refers to a ligand for the asialoglycoprotein receptor or a dendrimer thereof, eg, a compound described in the present invention.

Scheme 5 illustrates an exemplary preparation process for a Cas9 ribonucleoprotein in which RNA associates with Cas9 protein via electrostatic interactions, where n is an integer independently selected from 0 to 50 for each occurrence. .

Scheme 6 shows an exemplary synthesis of type L1-L10 linkers where Z is the peptide ppTG21, omitting hydrogen atoms in this peptide structure for clarity of illustration. One skilled in the art will readily appreciate that the peptide ppTG21 can be replaced with any small molecule, such as ASGPrL or an endosomal lysing agent, or the peptides described herein.

The present invention is also identical to that described herein except that one or more atoms are replaced with an atom having an atomic mass or mass number different from the atomic mass or mass number normally found in nature. Also included are isotopically-labeled compounds of the present invention. Examples of isotopes that can be incorporated into the compounds of the invention include hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine, iodine, and chlorine isotopes, eg, ² H, ³ H, ¹¹ C, ¹³ C, ¹⁴ C, ¹³ N, ¹⁵ N, ¹⁵ O, ¹⁷ O, ¹⁸ O, ³¹ P, ³² P, ³⁵ S, ¹⁸ F, ¹²³ I, ¹²⁵ I, and ³⁶ Cl.

Certain isotopically-labelled compounds of the present invention (eg, those labeled with ³ H and ¹⁴ C) are useful in compound and / or substrate tissue distribution assays. Tritium (ie, ³ H) and carbon-14 (ie, ¹⁴ C) isotopes are particularly preferred for their ease of preparation and detectability. In addition, substitution with heavy isotopes such as deuterium (ie ² H) may have certain therapeutic benefits (eg, increased in vivo half-life or reduced dosage required) due to higher metabolic stability. ) And therefore may be preferred in some situations. Positron emitting isotopes, such as ¹⁵ O, ¹³ N, ¹¹ C, and ¹⁸ F, are useful for positron emission tomography (PET) studies to examine substrate occupancy. The isotope-labeled compounds of the present invention are typically prepared by the following procedures similar to those disclosed in the following schemes and / or examples herein by replacing non-isotopically labeled reagents with isotope-labeled reagents. can do.

Endosome escape agent-containing composition Another aspect of the invention provides a composition comprising a compound described herein and an endosome escape agent described herein. In some embodiments of such compositions, the compound is co-incubated with the endosomal escape agent to form the composition.

  Another aspect of the invention is a composition comprising a ribonucleoprotein described herein (eg, an RNP comprising a site-specific modified polypeptide such as Cas9 RNP or Cpf1 RNP) and an endosomal escape agent described herein. Offer things. In some embodiments of such compositions, the ribonucleoprotein (eg, an RNP comprising a site-specific modified polypeptide such as Cas9 RNP or Cpf1 RNP) is co-incubated with the endosomal escape agent, and the composition is Form. In some embodiments, the ribonucleoprotein (eg, an RNP comprising a site-specific modified polypeptide such as Cas9 RNP or Cpf1 RNP) or the endosomal escape agent is conjugated to an antibody or fragment thereof. In some embodiments, the ribonucleoprotein (eg, an RNP comprising a site-specific modified polypeptide such as Cas9 RNP or Cpf1 RNP) is modified to include a glycosylation site. In some embodiments, the ribonucleoprotein (eg, an RNP comprising a site-specific modified polypeptide such as Cas9 RNP or Cpf1 RNP) is modified to include a transduction or translocation domain.

Pharmaceutical Compositions and Methods of Administration The compounds and compositions of the present invention are useful for the treatment of diseases, conditions and / or disorders. Accordingly, another embodiment of the present invention is a pharmaceutical composition comprising a therapeutically effective amount of a compound of the present invention, or an endosomal escape agent-containing composition, and a pharmaceutically acceptable excipient, diluent or carrier. is there. The compounds of the present invention (including the compositions and processes used herein) may also be used in the manufacture of a medicament for the therapeutic uses described herein.

  The pharmaceutical composition of the present invention may be in the form of a liquid solution (for example, an injection solution and an infusion solution). The preferred form depends on the intended mode of administration and therapeutic application. Conventional pharmaceutical compositions are in the form of solutions for injection or infusion, for example similar to those used for human passive immunization. One method of administration is parenteral (eg, intravenous, subcutaneous, intraperitoneal, intramuscular, intradermal, and intrasternal) in the form of a sterile injectable solution or oily suspension, or by infusion techniques It is. As will be appreciated by those skilled in the art, the route and / or method of administration will vary depending on the desired result. In preferred embodiments, the compound or composition is administered by intravenous infusion or injection. In another preferred embodiment, the compound or composition is administered by intramuscular or subcutaneous injection.

  Therapeutic pharmaceutical compositions are usually sterile and stable under the conditions of manufacture and storage.

  The pharmaceutical composition can be formulated as a solution, microemulsion, dispersion, or liposome. Sterile injectable solutions are prepared by incorporating the compound of the invention in the required amount, in an appropriate diluent, optionally with one or a combination of the above-listed components, followed by sterilization (eg, filter sterilization). Can do. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above. Such suspensions may be formulated according to the known art using suitable dispersions of wetting and suspending agents or other acceptable agents. The sterile injectable preparation may likewise be a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent, for example as a solution in 1,3-butanediol. Among the acceptable vehicles and solvents that can be employed are water, Ringer's solution, and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose any bland fixed oil can be employed including synthetic mono- or diglycerides. Furthermore, n-3 polyunsaturated fatty acids may find use in the preparation of injectables.

  In the case of sterile powders for the preparation of sterile injectable solutions, the preferred method of preparation is vacuum drying and lyophilization, from which the pre-sterilized filtered solution gives a powder of any further desired ingredients in addition to the active ingredient . The proper fluidity of a solution can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.

  Sustained absorption of an injectable pharmaceutical composition can be achieved by including in the pharmaceutical composition agents that delay absorption, for example, monostearate and gelatin, or the pharmaceutical composition is in a sustained absorption form, such as a depot, It can be provided by formulating into liposomes, polymer microspheres, polymer gels, and implants.

  Other methods of administration of the compounds of the invention described herein include skin patches that release the drug directly into the subject's skin. Such patches may contain a compound of the invention in a liquid solution optionally buffered, or may contain a compound of the invention dissolved and / or dispersed in an adhesive, or a compound of the invention dispersed in a polymer. May be included.

  The compound may be administered once, but may be administered multiple times. For example, the compound may be administered once a day to once every 6 months or more. The administration is three times a day, twice a day, once a day, once every two days, once every three days, once a week, once every two weeks, once a month, once every two months. Schedule once, every three months, once every six months, once a year, once every two years, etc.

  The compound can also be administered continuously with a minipump. The compound can be administered to the site of the affected area of the body or to a site remote from the site of the affected area of the body. The compound may be administered once, at least twice, or at least for a period of time until the disease is treated, alleviated or cured. The compound may generally be administered as long as the disease is present. The compound is usually administered as part of a pharmaceutical composition as described above.

  The pharmaceutical composition of the invention may comprise a therapeutically effective amount or a prophylactically effective amount of a compound of the invention. In preparing the pharmaceutical composition, the therapeutically effective amount of the compound contained in the pharmaceutical composition can be determined, for example, by the desired dosage volume and method (s) of administration, the nature and severity of the condition being treated, and the subject. Can be determined taking into account the age and size of the.

  An exemplary non-limiting dose range for administering a pharmaceutical composition of the invention to a subject is about 0.01 mg / kg to about 200 mg / kg (formula administered per kilogram (kg) of subject body weight (A) ) Or (B) expressed in milligrams (mg) of the compound), from about 0.1 mg / kg to about 100 mg / kg, from about 1.0 mg / kg to about 50 mg / kg, from about 5.0 mg / kg to about 20 mg / kg, or about 15 mg / kg. For the purposes of the present invention, an average human subject weighs about 70 kg. Ranges intermediate any of the dosages referred to herein, for example, about 0.02 mg / kg to 199 mg / kg are also intended to be part of the present invention. For example, it is intended to include a range of values using any combination of the values listed as upper and / or lower limits.

  The dosing regimen can be adjusted to give the subject the desired optimal response (eg, therapeutic or prophylactic response) by administering several divided doses over time, or the urgency of the treatment situation As indicated, the dose can be reduced or increased proportionally. It is especially advantageous to formulate parenteral pharmaceutical compositions in dosage unit form for ease of administration and uniformity of dosage.

  As used herein, dosage unit form refers to physically discrete units suitable as a single dose for the mammalian subject to be treated, each unit co-operating with a pharmaceutical carrier in need of the desired therapeutic effect. A predetermined amount of active compound calculated to occur. The specifications for the dosage unit form of the present invention are: (a) the specific properties of the compound or moiety and the specific therapeutic or prophylactic effect to be achieved, and (b) the formulation of the antibody for the treatment of hypersensitivity in an individual. It is determined by the limitations inherent in the technical field and depends directly on these.

  The liquid pharmaceutical composition of the present invention can be prepared as a unit dosage form. For example, a unit dose per vial may contain 1-1000 milliliters (ml) of different concentrations of the compound of formula (A) or (B). In other embodiments, the unit dose per vial is about 1 ml, 2 ml, 3 ml, 4 ml, 5 ml, 6 ml, 7 ml, 8 ml, 9 ml, 10 ml, 15 ml of different concentrations of the compound of formula (A) or (B), It can contain 20 ml, 30 ml, 40 ml, 50 ml, or 100 ml. If necessary, these formulations can be adjusted to the desired concentration by adding a sterile diluent to each vial. The liquid pharmaceutical compositions of the invention can also be prepared as unit dosage forms in sterile bags or containers, which are suitable for connection to an intravenous administration line or catheter.

  Another exemplary formulation is prepared by mixing a compound of the present invention and a carrier, diluent or excipient. Suitable carriers, diluents, and excipients are well known to those skilled in the art and include carbohydrates, waxes, water soluble and / or swellable polymers, hydrophilic or hydrophobic materials, gelatin, oils, solvents, water, etc. Contains materials. The particular unit, diluent or excipient used will depend upon the means and purpose for which the compound of the present invention is being applied. Solvents are generally selected based on solvents that are recognized by those skilled in the art as safe for administration to mammals (GRAS). In general, safe solvents are non-toxic aqueous solvents such as water and other non-toxic solvents that are water soluble or miscible. Suitable aqueous solvents include water, ethanol, propylene glycol, polyethylene glycol (eg, PEG400, PEG300), and the like, and mixtures thereof. The formulation may also include one or more buffering agents to give a sophisticated appearance of the drug (ie, a compound of the invention or pharmaceutical composition thereof) or to aid in the manufacture of a pharmaceutical product (ie, drug), Stabilizer, surfactant, wetting agent, lubricant, emulsifier, suspending agent, preservative, antioxidant, opacifier, glidant, processing aid, colorant, sweetener, fragrance, flavor Agents, and other known additives may be included.

  The formulation can be prepared using conventional dissolution and mixing procedures. For example, a drug substance (ie, a compound of the invention or a stabilized form of the compound (eg, a complex with a cyclodextrin derivative or other known complexing agent)) can be one or more in a suitable solvent. It is dissolved in the presence of the above excipients. The compounds of the present invention are usually formulated into pharmaceutical dosage forms to provide easily controllable doses of the drug and give the patient a sophisticated and easy to handle product.

  The pharmaceutical composition also includes solvates and hydrates of compounds of formula (A) or (B). The term “solvate” refers to a molecular complex of a compound of formula (A) or (B) (including pharmaceutically acceptable salts thereof) and one or more solvent molecules. Such solvent molecules are those commonly used in the pharmaceutical field that are known to be harmless to the recipient, such as water, ethanol, ethylene glycol, and the like. The term “hydrate” refers to the complex where the solvent molecule is water. The solvate and / or hydrate is preferably present in crystalline form. Other solvents, such as methanol, methyl t-butyl ether, ethyl acetate, methyl acetate, (S) -propylene glycol, (R) -propylene glycol, 1,4-butyne-diol, in the preparation of more desirable solvates, It can be used as an intermediate solvate. The crystalline form also exists as a complex with other harmless small molecules, such as L-phenylalanine, L-proline, L-pyroglutamic acid, as co-crystals or solvates or hydrates of co-crystal materials. In some cases. The solvates, hydrates, and co-crystal compounds may be prepared using the procedures described in PCT Publication No. WO 08/002824, or other procedures well known to those skilled in the art, incorporated herein by reference. Can be prepared.

  The pharmaceutical composition (or formulation) for application may be packaged in a variety of ways depending on the method used to administer the drug. Generally, an article for distribution includes a container in which the pharmaceutical formulation is deposited in a suitable form. Suitable containers are well known to those skilled in the art and include materials such as bottles (plastic and glass), sachets, ampoules, plastic bags, metal cylinders and the like. The container may also include a tamper-proof configuration to prevent inadvertent contact with the contents of the package. In addition, the container has a label that describes the contents of the container. The label may also include an appropriate warning.

Methods The compounds and compositions of the present application are useful for the treatment of various diseases or conditions. In some embodiments, the compounds and compositions described herein include hereditary angioedema, familial tyrosinemia type I, Alagille syndrome, alpha-1 antitrypsin deficiency, bile acid synthesis and metabolic disorders, Biliary atresia, cystic fibrosis liver disease, idiopathic neonatal hepatitis, mitochondrial hepatopathy, progressive familial intrahepatic cholestasis, primary sclerosing cholangitis, transthyretin amyloidosis, hemophilia, homozygosity Such as familial hypercholesterolemia, familial chylomicronemia, hyperlipidemia, steatohepatitis, nonalcoholic steatohepatitis (NASH), nonalcoholic fatty liver disease (NAFLD), type II diabetes Liver disease in a subject, including but not limited to hyperglycemia and diseases with abnormally high liver glucose production similar to type II diabetes Useful in the treatment of conditions or liver modulated disease or condition.

  The compounds and compositions of the present application are useful for the selective regulation of transcription of target DNA in a subject's liver cells, comprising administering an effective amount of a compound or composition described herein, , Hereditary angioedema, familial tyrosinemia type I, Alagille syndrome, alpha-1 antitrypsin deficiency, bile acid synthesis and metabolic disorders, biliary atresia, cystic fibrosis liver disease, idiopathic neonatal hepatitis, mitochondrial Liver disorder, progressive familial intrahepatic cholestasis, primary sclerosing cholangitis, transthyretin amyloidosis, hemophilia, homozygous familial hypercholesterolemia, familial chylomicronemia, hyperlipidemia , Steatohepatitis, nonalcoholic steatohepatitis (NASH), nonalcoholic fatty liver disease (NAFLD), hyperglycemia such as type II diabetes, and type II diabetes Is a disease or the like with similar abnormally high hepatic glucose production but not limited to, associated with liver disease or condition or liver modulated disease or condition, in a subject. In some embodiments, the target DNA is a PCSK9 gene.

  The compounds and compositions of the present application comprise hereditary angioedema, familial tyrosinemia type I, Alagille syndrome, alpha-1 antitrypsin, comprising administering an effective amount of a compound or composition described herein. Deficiency, bile acid synthesis and metabolic disorders, biliary atresia, cystic fibrosis liver disease, idiopathic neonatal hepatitis, mitochondrial hepatopathy, progressive familial intrahepatic cholestasis, primary sclerosing cholangitis, transthys Retin amyloidosis, hemophilia, homozygous familial hypercholesterolemia, familial chylomicronemia, hyperlipidemia, steatohepatitis, nonalcoholic steatohepatitis (NASH), nonalcoholic fatty liver disease (NAFLD), hyperglycemia such as type II diabetes, and diseases with abnormally high liver glucose production similar to type II diabetes, but not limited thereto Useful in editing of a nucleic acid molecule encoding a protein associated with liver disease or condition or liver modulated disease or condition, in a subject.

  The compounds and compositions of the present application comprise hereditary angioedema, familial tyrosinemia type I, Alagille syndrome, alpha-1 antitrypsin, comprising administering an effective amount of a compound or composition described herein. Deficiency, bile acid synthesis and metabolic disorders, biliary atresia, cystic fibrosis liver disease, idiopathic neonatal hepatitis, mitochondrial hepatopathy, progressive familial intrahepatic cholestasis, primary sclerosing cholangitis, transthys Retin amyloidosis, hemophilia, homozygous familial hypercholesterolemia, familial chylomicronemia, hyperlipidemia, steatohepatitis, nonalcoholic steatohepatitis (NASH), nonalcoholic fatty liver disease (NAFLD), hyperglycemia such as type II diabetes, and diseases with abnormally high liver glucose production similar to type II diabetes, but not limited thereto It is useful for the modulation of at least one gene product of the level associated with liver disease or condition or liver modulated disease or condition, in a subject expression. In some embodiments, the method modulates the level of low density lipoprotein (LDL). In some embodiments, the method modulates blood cholesterol levels in the subject. In some embodiments, the method reduces blood cholesterol levels in the subject.

  In some embodiments, the disease or condition targeted for the methods or uses described herein is hyperlipidemia, nonalcoholic steatohepatitis (NASH), or nonalcoholic fatty liver disease ( NAFLD). In some embodiments, the subject is a human.

  A composition comprising a ribonucleoprotein described herein (eg, an RNP comprising a site-specific modified polypeptide described herein such as Cas9 RNP or Cpf1 RNP) and an endosomal escape agent described herein is Useful for the treatment of disorders or diseases in a subject. In some embodiments, the subject is a human. In some embodiments, the disorder or disease is a blood disorder, cellular dysregulation or tumor disease and disorder, inflammation and immune related diseases, metabolic, liver, kidney, and protein diseases and disorders, muscle or skeletal diseases, Selected from, but not limited to, neurological and neurological diseases and disorders, and eye diseases and disorders. In some embodiments, the blood disorder or disease is anemia, naked lymphocyte syndrome, bleeding disorder, factor H and factor H-like 1, V, VIII, VII, X, XI, XII, XIIIA, or XIIIB deficiency, Fanconi anemia, hemophagocytic lymphohistiocytosis, hemophilia A or B, hemorrhagic disease, leukocyte deficiency and disorder, sickle cell anemia (HBB), and thalassemia. In some embodiments, the cellular dysregulation and tumor disease or disorder is selected from, but not limited to, B-cell non-Hodgkin lymphoma and leukemia. In some embodiments, the inflammation and immune related disease is selected from, but not limited to, AIDS, autoimmune lymphoproliferative syndrome, complex immune deficiency, HIV susceptibility or infection, severe complex immune deficiency . In some embodiments, the metabolic, liver, kidney, or protein disease is amyloid neuropathy, amyloidosis, cirrhosis, cystic fibrosis, glycogen storage disease, hepatic adenoma, liver failure, hepatic lipase deficiency, liver Selected from, but not limited to, blastoma, cancer and epithelial malignancy, medullary cystic kidney disease, phenylketonuria, polycystic kidney disease, and liver disease. In some embodiments, the muscle or skeletal disorder is selected from, but not limited to, Becker muscular dystrophy, Duchenne muscular dystrophy, Emery Dreyfus muscular dystrophy, scapulohumeral muscular dystrophy, muscular dystrophy, marble bone disease, muscle atrophy. It is not limited. In some embodiments, the neurological and neurological disease or disorder is ALS, Alzheimer's disease, autism, fragile X syndrome, Huntington's disease and disease-like disorder, Parkinson's disease, Rett syndrome, schizophrenia, secretase Selected from, but not limited to, related diseases, as well as trinucleotide repeat disorders. In some embodiments, the eye disease or disorder is selected from, but not limited to, age-related macular degeneration, cataracts, corneal opacity and dystrophy, congenital flat cornea, glaucoma, Leber's congenital cataract, and macular dystrophy .

  In some embodiments, the disease or disorder is neoplasia, age-related macular degeneration, CNS disorders (eg, schizophrenia or bipolar, Alzheimer's disease, Parkinson's disease, and autism), trinucleotide repeat disorders, Selected from, but not limited to, fragile X syndrome, secretase-related disease, prion-related disease, ALS, and drug addiction.

  A composition comprising a ribonucleoprotein described herein (eg, an RNP comprising a site-specific modified polypeptide such as Cas9 RNP or Cpf1 RNP) and an endosomal escape agent described herein is a cell function in a subject. Useful for adjustment. In some embodiments, intracellular signaling is PI3K / AKT signaling, ERK / MAPK signaling, glucocorticoid receptor signaling, axonal guidance signaling, ephrin receptor signaling, actin cytoskeleton signaling, Huntington's disease signaling, apoptosis signaling, B cell receptor signaling, leukocyte extravasation signaling, integrin signaling, acute phase response signaling, PTEN signaling, p53 signaling, aryl hydrocarbon receptor signaling, foreign body metabolism Signaling, SAPK / JNK signaling, PPAr / RXR signaling, NF-KB signaling, neuregulin signaling, Wnt and beta-catenin signaling, insulin receptor signaling, IL- Signal transduction, hepatic cholestasis, IGF-1 signaling, NRF2-mediated oxidative stress response, liver fibrosis / hepatic stellate cell activation, PPAR signaling, Fc epsilon RI signaling, G protein-coupled receptor signaling, inositol Phosphate metabolism, PDGF signaling, VEGF signaling, natural killer cell signaling, cell cycle: G1 / S checkpoint regulation, T cell receptor signaling, death receptor signaling, FGF signaling, GM-CSF signaling Signaling, amyotrophic lateral sclerosis signaling, JAK / Stat signaling, nicotinic acid and nicotinamide metabolism, chemokine signaling, IL-2 signaling, synaptic long-term inhibition, estrogen receptor signaling, protein ubiquitination pathway, I -10 signaling, VDR / RXR activation, TGF-beta signaling, Toll-like receptor signaling, p38 MAPK signaling, neurotrophin / TRK signaling, FXR / RXR activation, synaptic long-term potentiation, calcium signaling, EGF signaling, hypoxic signaling in the cardiovascular system, LPS / IL-1 mediated inhibition of RXR function, LXR / RXR activation, amyloid processing, IL-4 signaling, cell cycle: G2 / M DNA damage checkpoint regulation , Nitric oxide signaling in cardiovascular system, purine metabolism, cAMP-mediated signaling, mitochondrial dysfunction, Notch signaling, endoplasmic reticulum stress pathway, pyrimidine metabolism, parkinson signaling, heart and beta-adrenergic sig Signaling, glycolysis / gluconeogenesis, interferon signaling, sonic hedgehog signaling, glycerophospholipid metabolism, phospholipid degradation, tryptophan metabolism, lysine degradation, nucleotide excision repair pathway, starch and sucrose metabolism, amino sugar metabolism, arachidonic acid Metabolism, circadian rhythm signaling, coagulation system, dopamine receptor signaling, glutathione metabolism, glycerolipid metabolism, linoleic acid metabolism, methionine metabolism, pyruvate metabolism, arginine and proline metabolism, eicosanoid signaling, fructose and mannose metabolism, galactose Metabolism, biosynthesis of stilbene, coumarin and lignin, antigen presentation pathway, steroid biosynthesis, butanoic acid metabolism, citric acid cycle, fatty acid metabolism, glycerophospholipid metabolism, histidine metabolism, inositol metabolism, Metabolism of xenobiotics by tochrome P450, methane metabolism, phenylalanine metabolism, propanoic acid metabolism, selenoamino acid metabolism, sphingolipid metabolism, aminophosphonic acid metabolism, androgen and estrogen metabolism, ascorbic acid and aldaric acid metabolism, bile acid biosynthesis, cysteine metabolism , Fatty acid biosynthesis, glutamate receptor signaling, NRF2-mediated oxidative stress response, pentose phosphate pathway, pentose-glucuronic acid interconversion, retinol metabolism, riboflavin metabolism, tyrosine metabolism, ubiquinone biosynthesis, valine, leucine, and isoleucine degradation Selected from, but not limited to, metabolism of glycine, serine, and threonine, lysine degradation, pain / taste, pain, mitochondrial function, and developmental neurology.

  As described herein, a composition comprising a ribonucleoprotein (eg, an RNP comprising a site-specific modified polypeptide such as Cas9 RNP or Cpf1 RNP) and an endosomal escape agent is selective for transcription of target DNA in a subject. Useful for regulation, the DNA is associated with a disease or disorder as described herein. In some embodiments, the target DNA is associated with neoplasia and the DNA is PTEN, ATM, ATR, EGFR, ERBB2, ERBB3, ERBB4, Notch1, Notch2, Notch3, Notch4, AKT, AKT2, AKT3, HIF, HIFla, HIF3a, Met, HRG, Bcl2, PPARalpha, PPARgamma, WT1 (Wilms tumor), FGF receptor family members (5 members: 1, 2, 3, 4, 5), CDKN2a, APC, RB (retinoblastoma), MEN1, VHL, BRCA1, BRCA2, AR (androgen receptor), TSG101, IGF, IGF receptor, Igf1 (4 variant), Igf2 (3 variant), Igf1 receptor, Igf2 Receptor, Bax, Bcl2, Casper Family (9 members: 1,2,3,4,6,7,8,9,12), Kras, is selected from the Apc.

  In some embodiments, the target DNA is associated with age-related macular degeneration, and the DNA is selected from Abcr, Ccl2, Cc2, cp (ceruloplasmin), Timp3, cathepsin D, Vldrr, Ccr2 .

  In some embodiments, the target DNA is associated with a CNS disorder, such as neuregulin 1 (Nrg1), Erb4 (receptor for neuregulin), complexin 1 (Cplx1), Tph1 tryptophan hydroxy Rase, Tph2 tryptophan hydroxylase 2, Neulexin 1, GSK3, GSK3a, GSK3b, 5-HTT (Slc6a4), COMT, DRD (Drd1a), SLC6A3, DAOA, DTNBP1, Dao (Dao1), Mecp1, BGAP, BZAP , Neulexin 1, Fragile X (FMR2 (AFF2), FXR1, FXR2, Mglur5), E1, CHIP, UCH, UBB, Tau, LRP, PICALM, clusterin, PS1, S RL1, CR1, Vldlr, Uba1, Uba3, CHIP28 (Aqp1, aquaporin 1), Uchl1, Uchl3, APP, x- synuclein, is selected from DJ-1, LRRK2, Parkin, PINK1.

  In some embodiments, the target DNA is associated with a trinucleotide repeat disorder and the DNA is HTT (Huntington Dx), SBMA / SMAX1 / AR (Kennedy Dx), FXN / X25 (Friedrich ataxia) ), ATX3 (Mashad Joseph Dx), ATXN1 and ATXN2 (spinal cerebellar ataxia), DMPK (myotonic dystrophy), atrophin-1 and Atn1 (DRPLA Dx), CBP (Creb-BP-total instability), VLDLR (Alzheimer), Atxn7, Atxn10.

  In some embodiments, the target DNA is associated with fragile X syndrome, and the DNA is selected from FMR2, FXR1, FXR2, mGLUR5.

  In some embodiments, the target DNA is associated with a secretase-related disease, wherein the DNA is APH-1 (alpha and beta), preserinin (Psen1), nicastrin (Ncstn), PEN-2, Nos1, It is selected from Parp1, Nat1, and Nat2.

  In some embodiments, the target DNA is associated with a prion-related disease and the DNA is Prp.

  In some embodiments, the target DNA is associated with ALS, and the DNA is selected from SOD1, ALS2, STEX, FUS, TARDBP, VEGF (VEGF-a, VEGF-b, VEGF-c) The

  In some embodiments, the target DNA is associated with addiction, and the DNA is Prkce (alcohol), Drd2, Drd4, ABAT (alcohol), GRIA2, Grm5, Grin1, Htr1b, Grin2a, Drd3, Pdyn , Gria1 (alcohol).

  In some embodiments, the target DNA is associated with inflammation, and the DNA is IL-10, IL-1 (IL-1a, IL-1b), IL-13, IL-17 (IL- 17a (CTLA8), IL-17b, IL-17c, IL-17d, IL-17f), II-23, Cx3cr1, ptpn22, TNFa, NOD2 / CARD15 against IBD, IL-6, IL-12 (IL-12a, IL-12b), CTLA4, Cx3cl1.

  In some embodiments, the target DNA is associated with blood and coagulation diseases and disorders, and the DNA is CDAN1, CDA1, RPS19, DBA, PKLR, PK1, NT5C3, UMPH1, PSN1, RHAG, RH50A, NRAMP2, SPTB, ALAS2, ANH1, ASB, ABCB7, ABC7, ASAT, TAPBP, TPSN, TAP2, ABCB3, PSF2, RING11, MHC2TA, C2TA, RFX5, RFXAP, RFX5, TBXA2H, P2RX, P2RX1 MCFD2, F7, F10, F11, F12, HAF, F13A1, F13A, F13B, FANCA, FACA, FA1, FA, FAA, FAAP95, FAAP90, FLJ34064, FANCB, ANCC, FACC, BRCA2, FANCD1, FANCD2, FANCD, FACD, FAD, FANCE, FACE, FANCF, XRCC9, FANCG, BRIP1, BACH1, FANJ, PHF9, FANCL, FANCM, KIAA1596, PRF1, HPLM2, UNC13, UNC13, UNC13 HPLH3, HLH3, FHL3, F8, F8C, HEMA, F9, HEMB, PI, ATT, F5, ITGB2, CD18, LCAMB, LAD, EIF2B1, EIF2BA, EIF2B2, EIF2B3, EIF2B5, LVWM, BCH, B4, ECH2 It is selected from HBA2, HBB, HBD, LCRB, and HBA1.

  In some embodiments, the target DNA is associated with cellular dysregulation and tumor diseases and disorders, wherein the DNA is BCL7A, BCL7, TAL1, TCL5, SCL, TAL2, FLT3, NBS1, NBS, ZNFN1A1 , IK1, LYF1, HOXD4, HOX4B, BCR, CML, PHL, ALL, ARNT, KRAS2, RASK2, GMPS, AF10, ARHGEF12, LARG, KIAA0382, CALM, CLTH, CEBPA, CEBP, FLIC2, TTL, P3 , LPP, NPM1, NUP214, D9S46E, CAN, CAIN, RUNX1, CBFA2, AML1, WHSC1L1, NSD3, FLT3, AF1Q, NPM1, NUMA1, ZNF145, PLZF, PM , MYL, STAT5B, AF10, CALM, CLTH, ARL11, ARLTS1, P2RX7, P2X7, BCR, CML, PHL, ALL, GRAF, NF1, VRNF, WSS, NFNS, PTPN11, PTP2C, SHP2, NS1, BCL2, PR , BCL1, TCRA, GATA1, GF1, ERYF1, NFE1, ABL1, NQO1, DIA4, NMOR1, NUP214, D9S46E, CAN, CAIN.

  In some embodiments, the target DNA is associated with inflammation and immune related diseases and disorders, and the DNA is KIR3DL1, NKAT3, NKB1, AMB11, KIR3DS1, IFNG, CXCL12, SDF1, TNFRSF6, APT1, FAS , CD95, ALPS1A, IL2RG, SCIDX1, SCIDX, IMD4, CCL5, SCYA5, D17S136E, TCP228, IL10, CSIF, CMKBR2, CCR2, CMKBR5, CCCKR5 (CCR5), CD3E, CD3G, AICDA, AID2, HIDM, AID2, HIDM UNG, DGU, HIGM4, TNFSF5, CD40LG, HIGM1, IGM, FOXP3, IPEX, AIID, XPID, PIDX, TNFRSF14 , TACI, IL-10, IL-1 (IL-1a, IL-1b), IL-13, IL-17 (IL-17a (CTLA8), IL-17b, IL-17c, IL-17d, IL-17f ), II-23, Cx3cr1, ptpn22, TNFa, NOD2 / CARD15 to ILD, IL-6, IL-12 (IL-12a, IL-12b), CTLA4, Cx3cl1, JAK3, JAKL, DCLRE1C, ARTEMIS, SCIDA, RAG1 RAG2, ADA, PTPRC, CD45, LCA, IL7R, CD3D, T3D, IL2RG, SCIDX1, SCIDX, IMD4).

  In some embodiments, the target DNA is associated with metabolic, liver, kidney, and protein diseases and disorders, and the DNA is TTRPA1B, APOA1, APP, AAA, CVAP, AD1, GSN, FGA, LYZ, TTR, PA1B, KRT18, KRT8, CIRH1A, NAIC, TEX292, KIAA1988, CFTR, ABCC7, CF, MRP7, SLC2A2, GLUT2, G6PC, G6PT, G6PT1, GAA, LAMP2, LAMPB, AGL, GDE, SGL PYGL, PFKM, TCF1, HNF1A, MODY3, SCOD1, SCO1, LIPC, CTNNB1, PDGFRL, PDGRL, PRLTS, AXIN1, AXIN, CTNNB1, TP53, P53, LFS1, GF2R, MPRI, MET, CASP8, MCH5, UMOD, HNFJ, FJHN, MCKD2, ADMCKD2, PAH, PKU1, QDPR, DHPR, PTS, FCYT, PKHD1, ARPKD, PKD1, PKD2, PKD4, PKD4, PKD4, PKDTS, PKD4, PKDTS Selected from SEC63.

  In some embodiments, the target DNA is associated with muscle / skeletal diseases and disorders, and the DNA is DMD, BMD, MYF6, DMD, BMD, LMNA, LMN1, EMD2, FPLD, CMD1A, HGPS, LGMD1B, LMNA, LMN1, EMD2, FPLD, CMD1A, FSHMD1A, FSHD1A, FKRP, MDC1C, LGMD2I, LAMA2, LAMM, LARGE, KIAA0609, MDC1D, FCMD, TTID, MYOT, CAPN3G DMDA1, SCG3, SGCA, ADL, DAG2, LGMD2D, DMDA2, SGCB, LGMD2E, SGCD, SGD, LGMD2F, CMD1L, TCAP, LGMD2G, CMD N, TRIM32, HT2A, LGMD2H, FKRP, MDC1C, LGMD2I, TTN, CMD1G, TMD, LGMD2J, POMT1, CAV3, LGMD1C, SEPN1, SELN, RSMD1, PLEC1, PLTN, EBS1, RRP5, PGDL7 VBCH2, CLCN7, CLC7, OPTA2, OSTM1, GL, TCIRG1, TIRC7, OC116, OPTB1, VABP, VAPC, ALS8, SMN1, SMA1, SMA2, SMA3, SMA4, BSCL2, SPG17, GARS, SMADH, EMB2 It is selected from SMUBP2, CATF1, and SMARD1.

  In some embodiments, the target DNA is associated with neurological and neurological diseases and disorders, and the DNA is SOD1, ALS2, STEX, FUS, TARDBP, VEGF (VEGF-a, VEGF-b, VEGF-c, APP, AAA, CVAP, AD1, APOE, AD2, PSEN2, AD4, STM2, APBB2, FE65L1, NOS3, PLAU, URK, ACE, DCP1, ACE1, MPO, PACIP1, PAXIP1L, PTIP, A2M, BLMH BMH, PSEN1, AD3, Mecp2, BZRAP1, MDGA2, Sema5A, Neulexin 1, GLO1, MECP2, RTT, PPMX, MRX16, MRX79, NLGN3, NLGN4, KIAA1260, AUTSX2, FMR2, FX 1, FXR2, mGLUR5, HD, IT15, PRNP, PRIP, JPH3, JP3, HDL2, TBP, SCA17, NR4A2, NURR1, NOT, TINUR, SNCAIP, TBP, SCA17, SNCA, NACP, PARK1, PARK4, DJ1, PARK4, DJ7 LRRK2, PARK8, PINK1, PARK6, UCHL1, PARK5, SNCA, NACP, PARK1, PARK4, PRKN, PARK2, PDJ, DBH, NDUFV2, MECP2, RTT, PPMX, MRX16, MRX79, CDKL5, STK9, CDME5, STK9 MRX16, MRX79, x-synuclein, DJ-1, neuregulin 1 (Nrg1), Erb4 (receptor for neuregulin), complex 1 (Cplx1), Tph1 tryptophan hydroxylase, Tph2, tryptophan hydroxylase 2, neulexin 1, GSK3, GSK3a, GSK3b, 5-HTT (Slc6a4), COMT, DRD (Drd1a), SLC6A3, DAOA, DTNBP1, Da1Do ), APH-1 (alpha and beta), presenilin (Psenl), nicastrin, (Ncstn), PEN-2, Nos1, Parp1, Nat1, Nat2, HTT (Huntington Dx), SBMA / SMAX1 / AR (Kennedy Dx), FXN / X25 (Friedrich ataxia), ATX3 (Mashad Joseph Dx), ATXN1 and ATXN2 (spinal cerebellar ataxia), DMPK (myotonic dystrophy), atrophin-1 and And Atn1 (DRPLA Dx), CBP (Creb-BP-total instability), VLDLR (Alzheimer), Atxn7, Atxn10.

  In some embodiments, the target DNA is associated with eye diseases and disorders, the DNA being Abcr, Ccl2, Cc2, cp (ceruloplasmin), Timp3, cathepsin D, Vldrr, Ccr2, CRYAA, CRYA1 , CRYBB2, CRYB2, PITX3, BFSP2, CP49, CP47, CRYAA, CRYA1, PAX6, AN2, MGDA, CRYBA1, CRYB1, CRYGC, CRYG3, CCL, LIM2, MP19, CRYGD, CRYG4, BFSP2, C49G , HSF4, CTM, MIP, AQP0, CRYAB, CRYA2, CTPP2, CRYBB1, CRYGD, CRYG4, CRYBB2, CRYB2, CRYGC, CRYG3, CCL, CRYAA, CR A1, GJA8, CX50, CAE1, GJA3, CX46, CZP3, CAE3, CCM1, CAM, KRIT1, APOA1, TGFBI, CSD2, CDGG1, CSD, BIGH3, CDG2, TACSTD2, TROP2, M1R1, PSX KTCN, COL8A2, FECD, PPCD2, PIP5K3, CFD, KERA, CNA2, MYOC, TIGR, GLC1A, JOAG, GPOA, OPTN, GLC1E, FIP2, HYPL, NRP, CYP1B1, GLC3A, OPG1, APG1A CRB1, RP12, CRX, CORD2, CRD, RPGRIP1, LCA6, CORD9, RPE65, RP20, AIPL1, LCA4, GUCY D, GUC2D, LCA1, CORD6, RDH12, LCA3, ELOVL4, ADMD, STGD2, STGD3, RDS, RP7, PRPH2, PRPH, AVMD, selected from AOFMD, VMD2.

  As described herein, a composition comprising a ribonucleoprotein (eg, an RNP comprising a site-specific modified polypeptide such as Cas9 RNP or Cpf1 RNP) and an endosomal escape agent is selective for target DNA transcription in a subject. Useful for regulation, the DNA is associated with cellular function as described herein.

  In some embodiments, the target DNA is associated with PI3K / AKT signaling and the DNA is PRKCE, ITGAM, ITGA5, IRAK1, PRKAA2, EIF2AK2, PTEN, EIF4E, PRKCZ, GRK6, MAPK1, TSC1 , PLK1, AKT2, IKBKB, PIK3CA, CDK8, CDKN1B, NFKB2, BCL2, PIK3CB, PPP2R1A, MAPK8, BCL2L1, MAPK3, TSC2, ITGA1, KRAS, EIF4EBP1, RELA, PRK9, RELA, PRK9, RELA, PRK9 , ITGB7, YWHAZ, ILK, TP53, RAF1, IKBKG, RELB, DYRK1A, CDKN1A, ITGB1, MAP2K , JAK1, AKT1, JAK2, PIK3R1, CHUK, PDPK1, PPP2R5C, CTNNB1, MAP2K1, NFKB1, PAK3, ITGB3, CCND1, GSK3A, FRAP1, SFN, ITGA2, TTK, CSK , RPS6KB1.

  In some embodiments, the target DNA is associated with ERK / MAPK signaling, and the DNA is PRKCE, ITGAM, ITGA5, HSPB1, IRAK1, PRKAA2, EIF2AK2, RAC1, RAP1A, TLN1, EIF4E, ELK1 , GRK6, MAPK1, RAC2, PLK1, AKT2, PIK3CA, CDK8, CREB1, PRKCI, PTK2, FOS, RPS6KA4, PIK3CB, PPP2R1A, PIK3C3, MAPK8, MAPK3, ITGA1, ETS1, ETS1, ETS1, ETS1, PTS , MAPK9, SRC, CDK2, PPP2CA, PIM1, PIK3C2A, ITGB7, YWHAZ, PPP1CC, KSR1, PXN, AF1, FYN, DYRK1A, ITGB1, MAP2K2, PAK4, PIK3R1, STAT3, PPP2R5C, MAP2K1, PAK3, ITGB3, ESR1, ITGA2, MYC, TTK, CSNK1A1, CRKL, BRK, ATK4, PRK, ATFS4, PRK, ATFS4 The

  In some embodiments, the target DNA is associated with glucocorticoid receptor signaling, and the DNA is RAC1, TAF4B, EP300, SMAD2, TRAF6, PCAF, ELK1, MAPK1, SMAD3, AKT2, IKBKB, NCOR2, UBE2I, PIK3CA, CREB1, FOS, HSPA5, NFKB2, BCL2, MAP3K14, STAT5B, PIK3CB, PIK3C3, MAPK8, BCL2L1, MAPK3, TSC22D3, MAPK10, NRIP1, KRAP9, NRIP1, KRAS, MMAP10 NR3C1, PIK3C2A, CDKN1C, TRAF2, SERPINE1, NCOA3, MAPK14, TNF, RAF1, IKBKG MAP3K7, CREBBP, CDKN1A, MAP2K2, JAK1, IL8, NCOA2, AKT1, JAK2, PIK3R1, CHUK, STAT3, MAP2K1, NFKB1, TGFBR1, ESR1, SMAD4, CEBPB, JKT3, CEBPB, JKT1 Selected from HSP90AA1.

  In some embodiments, the target DNA is associated with axonal guidance signaling and the DNA is PRKCE, ITGAM, ROCK1, ITGA5, CXCR4, ADAM12, IGF1, RAC1, RAP1A, E1F4E, PRKCZ, NRP1 , NTRK2, ARHGEF7, SMO, ROCK2, MAPK1, PGF, RAC2, PTPN11, GNAS, AKT2, PIK3CA, ERBB2, PRKCI, PTK2, CFL1, GNAQ, PIK3CB, CXCL12, PIK3C3, WNT11, PRD3M3, WNT11, PRD , KRAS, RHOA, PRKCD, PIK3C2A, ITGB7, GLI2, PXN, VASP, RAF1, FYN, ITGB1, MAP2K2, PAK4, A AM17, AKT1, PIK3R1, GLI1, WNT5A, ADAM10, MAP2K1, PAK3, ITGB3, CDC42, VEGFA, ITGA2, EPHA8, CRKL, selected from RND1, GSK3B, AKT3, PRKCA.

  In some embodiments, the target DNA is associated with ephrin receptor signaling, and the DNA is PRKCE, ITGAM, ROCK1, ITGA5, CXCR4, IRAK1, PRKAA2, EIF2AK2, RAC1, RAP1A, GRK6, ROCK2 , MAPK1, PGF, RAC2, PTPN11, GNAS, PLK1, AKT2, DOK1, CDK8, CREB1, PTK2, CFL1, GNAQ, MAP3K14, CXCL12, MAPK8, GNB2L1, ABL1, MAPK3, ITGA1, PRA9K , SRC, CDK2, PIM1, ITGB7, PXN, RAF1, FYN, DYRK1A, ITGB1, MAP2K2, PAK4, AKT1, JAK2, STA 3, ADAM10, MAP2K1, PAK3, ITGB3, CDC42, VEGFA, ITGA2, EPHA8, TTK, CSNK1A1, CRKL, is selected from the BRAF, PTPN13, ATF4, AKT3, SGK.

  In some embodiments, the target DNA is associated with actin cytoskeleton signaling and the DNA is ACTN4, PRKCE, ITGAM, ROCK1, ITGA5, IRAK1, PRKAA2, EIF2AK2, RAC1, INS, ARGGEF7, GRK6. , ROCK2, MAPK1, RAC2, PLK1, AKT2, PIK3CA, CDK8, PTK2, CFL1, PIK3CB, MYH9, DIAPH1, PIK3C3, MAPK8, F2R, MAPK3, SLC9A1, ITGA1, KRAS, RHOAK, PR , PIK3C2A, ITGB7, PPP1CC, PXN, VIL2, RAF1, GSN, DYRK1A, ITGB1, MAP2K2, PAK4, PIP5K A, PIK3R1, MAP2K1, PAK3, ITGB3, CDC42, APC, ITGA2, TTK, CSNK1A1, CRKL, is selected from the BRAF, VAV3, SGK.

  In some embodiments, the target DNA is associated with Huntington's disease signaling, and the DNA is PRKCE, IGF1, EP300, RCOR1, PRKCZ, HDAC4, TGM2, MAPK1, CAPNS1, AKT2, EGFR, NCOR2, SP1, CAPN2, PIK3CA, HDAC5, CREB1, PRKC1, HSPA5, REST, GNAQ, PIK3CB, PIK3C3, MAPK8, IGF1R, PRKD1, GNB2L1, BCL2L1, CAPN1, MAPK3, CASP8, HDAC11, KAP9, HDAC9 PIK3C2A, HDAC3, TP53, CASP9, CREBBP, AKT1, PIK3R1, PDPK1, CASP1, APAF1, FR P1, CASP2, JUN, BAX, ATF4, AKT3, PRKCA, CLTC, is selected from SGK, HDAC6, CASP3.

  In some embodiments, the target DNA is associated with apoptosis signaling, and the DNA is PRKCE, ROCK1, BID, IRAK1, PRKAA2, EIF2AK2, BAK1, BIRC4, GRK6, MAPK1, CAPNS1, PLK1, AKT2 , IKBKB, CAPN2, CDK8, FAS, NFKB2, BCL2, MAP3K14, MAPK8, BCL2L1, CAPN1, MAPK3, CASP8, KRAS, RELA, PRKCD, PRKAA1, MAPK9, CDK2, PIM1, GTP1, GTP1, SP5 , DYRK1A, MAP2K2, CHUK, APAF1, MAP2K1, NFKB1, PAK3, LMNA, CASP2, BIRC2, TTK, CSNK1A , BRAF, BAX, PRKCA, is selected from SGK, CASP3, BIRC3, PARP1.

  In some embodiments, the target DNA is associated with B cell receptor signaling, and the DNA is RAC1, PTEN, LYN, ELK1, MAPK1, RAC2, PTPN11, AKT2, IKBKB, PIK3CA, CREB1, SYK, NFKB2, CAMK2A, MAP3K14, PIK3CB, PIK3C3, MAPK8, BCL2L1, ABL1, MAPK3, ETS1, KRAS, MAPK13, RELA, PTPN6, MAPK9, EGR1, PIK3C1A, B14 AKT1, PIK3R1, CHUK, MAP2K1, NFKB1, CDC42, GSK3A, FRAP1, BCL6, BCL10, JUN, GSK3B, ATF4, A T3, VAV3, is selected from RPS6KB1.

  In some embodiments, the target DNA is associated with leukocyte extravasation signaling, wherein the DNA is ACTN4, CD44, PRKCE, ITGAM, ROCK1, CXCR4, CYBA, RAC1, RAP1A, PRKCZ, ROCK2, RAC2, PTPN11, MMP14, PIK3CA, PRKCI, PTK2, PIK3CB, CXCL12, PIK3C3, MAPK8, PRKD1, ABL1, MAPK10, CYBB, MAPK13, RHOA, PRKCD, MAPK9, SRC, PIK3X2K, VTK ITGB1, MAP2K2, CTNND1, PIK3R1, CTNNB1, CLDN1, CDC42, F11R, ITK, CRKL, VAV3, CTTN, PRK It is selected from A, MMP1, MMP9.

  In some embodiments, the target DNA is associated with integrin signaling, and the DNA is ACTN4, ITGAM, ROCK1, ITGA5, RAC1, PTEN, RAP1A, TLN1, ARHGEF7, MAPK1, RAC2, CAPNS1, AKT2 CAPN2, PIK3CA, PTK2, PIK3CB, PIK3C3, MAPK8, CAV1, CAPN1, ABL1, MAPK3, ITGA1, KRAS, RHOA, SRC, PIK3C2A, ITGB7, PPP1CC, ILK, PXN, VASP, RAP4K , AKT1, PIK3R1, TNK2, MAP2K1, PAK3, ITGB3, CDC42, RND3, ITGA2, CRKL, BRAF, GSK3B, AK 3 is selected from.

  In some embodiments, the target DNA is associated with acute phase response signaling, and the DNA is IRAK1, SOD2, MYD88, TRAF6, ELK1, MAPK1, PTPN11, AKT2, IKBKB, PIK3CA, FOS, NFKB2 , MAP3K14, PIK3CB, MAPK8, RIPK1, MAPK3, IL6ST, KRAS, MAPK13, IL6R, RELA, SOCS1, MAPK9, FTL, NR3C1, TRAF2, SERPINE1, MAPK14, TNF, RAF1, PDK1, TIF2, KDK1, T , JAK2, PIK3R1, CHUK, STAT3, MAP2K1, NFKB1, FRAP1, CEBPB, JUN, AKT3, IL1R1, IL6 It is.

  In some embodiments, the target DNA is associated with PTEN signaling, and the DNA is ITGAM, ITGA5, RAC1, PTEN, PRKCZ, BCL2L11, MAPK1, RAC2, AKT2, EGFR, IKBKB, CBL, PIK3CA. , CDKN1B, PTK2, NFKB2, BCL2, PIK3CB, BCL2L1, MAPK3, ITGA1, KRAS, ITGB7, ILK, PDGFRB, INSR, RAF1, IKBKG, CASP9, CDKN1A, ITGB1, MAP1PI, MAP2K2, AK1 , NFKB1, ITGB3, CDC42, CCND1, GSK3A, ITGA2, GSK3B, AKT3, FOXO1, CASP3, RPS6KB It is selected from.

  In some embodiments, the target DNA is associated with p53 signaling, and the DNA is PTEN, EP300, BBC3, PCAF, FASN, BRCA1, GADD45A, BIRC5, AKT2, PIK3CA, CHEK1, TP53INP1, BCL2, PIK3CB, PIK3C3, MAPK8, THBS1, ATR, BCL2L1, E2F1, PMAIP1, CHEK2, TNFRSF10B, TP73, RB1, HDAC9, CDK2, PIK3C2A, MAPK14, TP53, LRDD, CDKNR CTNNB1, SIRT1, CCND1, PRKDC, ATM, SFN, CDKN2A, JUN, SNAI2, GSK3B, BAX, AKT3 Are al selected.

  In some embodiments, the target DNA is associated with aryl hydrocarbon receptor signaling, and the DNA is HSPB1, EP300, FASN, TGM2, RXRA, MAPK1, NQO1, NCOR2, SP1, ARNT, CDKN1B , FOS, CHEK1, SMARCA4, NFKB2, MAPK8, ALDH1A1, ATR, E2F1, MAPK3, NRIP1, CHEK2, RELA, TP73, GSTP1, RB1, SRC, CDK2, AHR, NFE2L2, NCOA3, TP53A, NCOA3, TP53A , NFKB1, CCND1, ATM, ESR1, CDKN2A, MYC, JUN, ESR2, BAX, IL6, CYP1B1, and HSP90AA1.

  In some embodiments, the target DNA is associated with xenobiotic metabolic signaling, and the DNA is PRKCE, EP300, PRKCZ, RXRA, MAPK1, NQO1, NCOR2, PIK3CA, ARNT, PRKCI, NFKB2, CAMK2A, PIK3CB, PPP2R1A, PIK3C3, MAPK8, PRKD1, ALDH1A1, MAPK3, NRIP1, KRAS, MAPK13, PRKCD, GSTP1, MAPK9, NOS2A, ABCB1, AHR, PPP2CA, FTL, NFE2 MAP2K2, PIK3R1, PPP2R5C, MAP2K1, NFKB1, KEAP1, PRKCA, EIF2AK3, IL6, C P1B1, is selected from HSP90AA1.

  In some embodiments, the target DNA is associated with SAPK / JNK signaling, and the DNA is PRKCE, IRAK1, PRKAA2, EIF2AK2, RAC1, ELK1, GRK6, MAPK1, GADD45A, RAC2, PLK1, AKT2. , PIK3CA, FADD, CDK8, PIK3CB, PIK3C3, MAPK8, RIPK1, GNB2L1, IRS1, MAPK3, MAPK10, DAXX, KRAS, PRKCD, PRKAA1, MAPK9, CDK2, PIM1, FIK2C3A2 , PIK3R1, MAP2K1, PAK3, CDC42, JUN, TTK, CSNK1A1, CRKL, BRAF, SGK.

  In some embodiments, the target DNA is associated with PPAr / RXR signaling, the DNA is PRKAA2, EP300, INS, SMAD2, TRAF6, PPARA, FASN, RXRA, MAPK1, SMAD3, GNAS, IKBKB , NCOR2, ABCA1, GNAQ, NFKB2, MAP3K14, STAT5B, MAPK8, IRS1, MAPK3, KRAS, RELA, PRKAA1, PPARGC1A, NCOA3, MAPK1, INSR, RAF1, IKBKG, RELB, MAPK, RELB, MAPK , NFKB1, TGFBR1, SMAD4, JUN, IL1R1, PRKCA, IL6, HSP90AA1, and ADIPOQ.

  In some embodiments, the target DNA is associated with NF-KB signaling, and the DNA is IRAK1, EIF2AK2, EP300, INS, MYD88, PRKCZ, TRAF6, TBK1, AKT2, EGFR, IKBKB, PIK3CA. , BTRC, NFKB2, MAP3K14, PIK3CB, PIK3C3, MAPK8, RIPK1, HDAC2, KRAS, RELA, PIK3C2A, TRAF2, TLR4, PDGFRB, TNF, INSR, LCK, IKBKG, RELB, MAP3K7, RELB, MAP3K7 , NFKB1, TLR2, BCL10, GSK3B, AKT3, TNFAIP3, and IL1R1.

  In some embodiments, the target DNA is associated with neuregulin signaling, and the DNA is ERBB4, PRKCE, ITGAM, ITGA5, PTEN, PRKCZ, ELK1, MAPK1, PTPN11, AKT2, EGFR, ERBB2, PRKCI, CDKN1B, STAT5B, PRKD1, MAPK3, ITGA1, KRAS, PRKCD, STAT5A, SRC, ITGB7, RAF1, ITGB1, MAP2K2, ADAM17, AKT1, PIK3R1, PDPK1, ITGB3, ITGP3E, ITGB1, ITGB3 It is selected from NRG1, CRKL, AKT3, PRKCA, HSP90AA1, and RPS6KB1.

  In some embodiments, the target DNA is associated with Wnt and beta-catenin signaling, and the DNA is CD44, EP300, LRP6, DVL3, CSNK1E, GJA1, SMO, AKT2, PIN1, CDH1, BTRC, GNAQ, MARK2, PPP2R1A, WNT11, SRC, DKK1, PPP2CA, SOX6, SFRP2, ILK, LEF1, SOX9, TP53, MAP3K7, CREBBP, TCF7L2, AKT1, PPP2R5C, WNT5A, LRP5T, NPT5A, LRP5 It is selected from APC, CDKN2A, MYC, CSNK1A1, GSK3B, AKT3 and SOX2.

  In some embodiments, the target DNA is associated with insulin receptor signaling and the DNA is PTEN, INS, EIF4E, PTPN1, PRKCZ, MAPK1, TSC1, PTPN11, AKT2, CBL, PIK3CA, PRKCI , PIK3CB, PIK3C3, MAPK8, IRS1, MAPK3, TSC2, KRAS, EIF4EBP1, SLC2A4, PIK3C2A, PPP1CC, INSR, RAF1, FYN, MAP2K2, JAK1, AKT1, JK1, AKT1, JAK2, PIK3, PIK3 , AKT3, FOXO1, SGK, RPS6KB1.

  In some embodiments, the target DNA is associated with IL-6 signaling and the DNA is HSPB1, TRAF6, MAPKAPK2, ELK1, MAPK1, PTPN11, IKBKB, FOS, NFKB2, MAP3K14, MAPK8, MAPK3 , MAPK10, IL6ST, KRAS, MAPK13, IL6R, RELA, SOCS1, MAPK9, ABCB1, TRAF2, MAPK14, TNF, RAF1, IKBKG, RELB, MAP3K7, MAP2K2, IL8, JAK2, PUK, STAT3 , IL1R1, SRF, and IL6.

  In some embodiments, the target DNA is associated with hepatic cholestasis, and the DNA is PRKCE, IRAK1, INS, MYD88, PRKCZ, TRAF6, PPARA, RXRA, IKBKB, PRKCI, NFKB2, MAP3K14, MAPK8, PRKD1, MAPK10, RELA, PRKCD, MAPK9, ABCB1, TRAF2, TLR4, TNF, INSR, IKBKG, RELB, MAP3K7, IL8, CHUK, NR1H2, TJP2, NFKB1, ESR1, SRFK, ESR1, SREFK Selected from IL6.

  In some embodiments, the target DNA is associated with IGF-1 signaling and the DNA is IGF1, PRKCZ, ELK1, MAPK1, PTPN11, NEDD4, AKT2, PIK3CA, PRKCI, PTK2, FOS, PIK3CB , PIK3C3, MAPK8, IGF1R, IRS1, MAPK3, IGFBP7, KRAS, PIK3C2A, YWHAZ, PXN, RAF1, CASP9, MAP2K2, AKT1, PIK3R1, PDPK1, MAP2K1, TGFBP2S, IGFBP2 , RPS6KB1.

  In some embodiments, the target DNA is associated with an NRF2-mediated oxidative stress response, and the DNA is PRKCE, EP300, SOD2, PRKCZ, MAPK1, SQSTM1, NQO1, PIK3CA, PRKCI, FOS, PIK3CB, PIK3C3 , MAPK8, PRKD1, MAPK3, KRAS, PRKCD, GSTP1, MAPK9, FTL, NFE2L2, PIK3C2A, MAPK14, RAF1, MAP3K7, CREBBP, MAP2K2, AKT1, PIK3R1, PIMAP3R1, PIMAP3R1, PIMAP3K , HSP90AA1.

  In some embodiments, the target DNA is associated with hepatic fibrosis / hepatic stellate cell activation, and the DNA is EDN1, IGF1, KDR, FLT1, SMAD2, FGFR1, MET, PGF, SMAD3, EGFR, FAS, CSF1, NFKB2, BCL2, MYH9, IGF1R, IL6R, RELA, TLR4, PDGFRB, TNF, RELB, IL8, PDGFRA, NFKB1, TGFBR1, SMAD4, VEGFA, BAX, IL1ST1, CCL2, IL1R1, CCL2 It is selected from IL6, CTGF, and MMP9.

  In some embodiments, the target DNA is associated with PPAR signaling and the DNA is EP300, INS, TRAF6, PPARA, RXRA, MAPK1, IKBKB, NCOR2, FOS, NFKB2, MAP3K14, STAT5B, MAPK3 NRIP1, KRAS, PPARG, RELA, STAT5A, TRAF2, PPARGC1A, PDGFRB, TNF, INSR, RAF1, IKBKG, RELB, MAP3K7, CREBBP, MAP2K2, CHUK, PDGFRA, MAP2K1, RNF1, J .

  In some embodiments, the target DNA is associated with Fc epsilon RI signaling and the DNA is PRKCE, RAC1, PRKCZ, LYN, MAPK1, RAC2, PTPN11, AKT2, PIK3CA, SYK, PRKCI, PIK3CB. , PIK3C3, MAPK8, PRKD1, MAPK3, MAPK10, KRAS, MAPK13, PRKCD, MAPK9, PIK3C2A, BTK, MAPK14, TNF, RAF1, FYN, MAP2K2, AKT1, PIK3R3, PDPK1, MAPK1, AP2 .

  In some embodiments, the target DNA is associated with G protein-coupled receptor signaling, and the DNA is PRKCE, RAP1A, RGS16, MAPK1, GNAS, AKT2, IKBKB, PIK3CA, CREB1, GNAQ, NFKB2 , CAMK2A, PIK3CB, PIK3C3, MAPK3, KRAS, RELA, SRC, PIK3C2A, RAF1, IKBKG, RELB, FYN, MAP2K2, AKT1, PIK3R1, CHUK, PDPK1, STAT3, MAP3K, MAP3T Is done.

  In some embodiments, the target DNA is associated with inositol phosphate metabolism, and the DNA is PRKCE, IRAK1, PRKAA2, EIF2AK2, PTEN, GRK6, MAPK1, PLK1, AKT2, PIK3CA, CDK8, PIK3CB, PIK3C3, MAPK8, MAPK3, PRKCD, PRKAA1, MAPK9, CDK2, PIM1, PIK3C2A, DYRK1A, MAP2K2, PIP5K1A, PIK3R1, MAP2K1, PAK3, ATM, TTK, CSNK1SG, RANK1A1, B

  In some embodiments, the target DNA is associated with PDGF signaling, and the DNA is EIF2AK2, ELK1, ABL2, MAPK1, PIK3CA, FOS, PIK3CB, PIK3C3, MAPK8, CAV1, ABL1, MAPK3, KRAS , SRC, PIK3C2A, PDGFRB, RAF1, MAP2K2, JAK1, JAK2, PIK3R1, PDGFRA, STAT3, SPHK1, MAP2K1, MYC, JUN, CRKL, PRKCA, SRF, STAT1, SPHK2.

  In some embodiments, the target DNA is associated with VEGF signaling and the DNA is ACTN4, ROCK1, KDR, FLT1, ROCK2, MAPK1, PGF, AKT2, PIK3CA, ARNT, PTK2, BCL2, PIK3CB , PIK3C3, BCL2L1, MAPK3, KRAS, HIF1A, NOS3, PIK3C2A, PXN, RAF1, MAP2K2, ELAVL1, AKT1, PIK3R1, MAP2K1, SFN, VEGFA, AKT3, FOXO1, and POXO1.

  In some embodiments, the target DNA is associated with natural killer cell signaling and the DNA is PRKCE, RAC1, PRKCZ, MAPK1, RAC2, PTPN11, KIR2DL3, AKT2, PIK3CA, SYK, PRKCI, PIK3CB. , PIK3C3, PRKD1, MAPK3, KRAS, PRKCD, PTPN6, PIK3C2A, LCK, RAF1, FYN, MAP2K2, PAK4, AKT1, PIK3R1, MAP2K1, PAK3, AKT3, VAV3, PRKCA.

  In some embodiments, the target DNA is associated with cell cycle: G1 / S checkpoint modulation and the DNA is HDAC4, SMAD3, SUV39H1, HDAC5, CDKN1B, BTRC, ATR, ABL1, E2F1, HDAC2 , HDAC7A, RB1, HDAC11, HDAC9, CDK2, E2F2, HDAC3, TP53, CDKN1A, CCND1, E2F4, ATM, RBL2, SMAD4, CDKND2A, MYC, NRG1, GSK3B, RBL1, and HDAC6.

  In some embodiments, the target DNA is associated with T cell receptor signaling, and the DNA is RAC1, ELK1, MAPK1, IKBKB, CBL, PIK3CA, FOS, NFKB2, PIK3CB, PIK3C3, MAPK8, It is selected from MAPK3, KRAS, RELA, PIK3C2A, BTK, LCK, RAF1, IKBKG, RELB, FYN, MAP2K2, PIK3R1, CHUK, MAP2K1, NFKB1, ITK, BCL10, JUN, and VAV3.

  In some embodiments, the target DNA is associated with cell death receptor signaling, and the DNA is CRADD, HSPB1, BID, BIRC4, TBK1, IKBKB, FADD, FAS, NFKB2, BCL2, MAP3K14, MAPK8, RIPK1, CASP8, DAXX, TNFRSF10B, RELA, TRAF2, TNF, IKBKG, RELB, CASP9, CHUK, APAF1, NFKB1, CASP2, BIRC2, CASP3, BIRC3.

  In some embodiments, the target DNA is associated with FGF signaling, and the DNA is RAC1, FGFR1, MET, MAPKAPK2, MAPK1, PTPN11, AKT2, PIK3CA, CREB1, PIK3CB, PIK3C3, MAPK8, MAPK3. , MAPK13, PTPN6, PIK3C2A, MAPK14, RAF1, AKT1, PIK3R1, STAT3, MAP2K1, FGFR4, CRKL, ATF4, AKT3, PRKCA, HGF.

  In some embodiments, the target DNA is associated with GM-CSF signaling and the DNA is LYN, ELK1, MAPK1, PTPN11, AKT2, PIK3CA, CAMK2A, STAT5B, PIK3CB, PIK3C3, GNB2L1, BCL2L1 , MAPK3, ETS1, KRAS, RUNX1, PIM1, PIK3C2A, RAF1, MAP2K2, AKT1, JAK2, PIK3R1, STAT3, MAP2K1, CCND1, AKT3, and STAT1.

  In some embodiments, the target DNA is associated with amyotrophic lateral sclerosis signaling, and the DNA is BID, IGF1, RAC1, BIRC4, PGF, CAPNS1, CAPN2, PIK3CA, BCL2, PIK3CB. , PIK3C3, BCL2L1, CAPN1, PIK3C2A, TP53, CASP9, PIK3R1, RAB5A, CASP1, APAF1, VEGFA, BIRC2, BAX, AKT3, CASP3, BIRC3.

  In some embodiments, the target DNA is associated with JAK / Stat signaling, and the DNA is PTPN1, MAPK1, PTPN11, AKT2, PIK3CA, STAT5B, PIK3CB, PIK3C3, MAPK3, KRAS, SOCS1, STAT5A , PTPN6, PIK3C2A, RAF1, CDKN1A, MAP2K2, JAK1, AKT1, JAK2, PIK3R1, STAT3, MAP2K1, FRAP1, AKT3, and STAT1.

  In some embodiments, the target DNA is associated with nicotinic acid and nicotinamide metabolism, and the DNA is PRKCE, IRAK1, PRKAA2, EIF2AK2, GRK6, MAPK1, PLK1, AKT2, CDK8, MAPK8, MAPK3, It is selected from PRKCD, PRKAA1, PBEF1, MAPK9, CDK2, PIM1, DYRK1A, MAP2K2, MAP2K1, PAK3, NT5E, TTK, CSNK1A1, BRAF, and SGK.

In some embodiments, the target DNA is associated with chemokine signaling, and the DNA is CXCR4, ROCK2, MAPK1, PTK2, FOS, CFL1, GNAQ, CAMK2A, CXCL12, MAPK8, MAPK3, KRAS, MAPK13
, RHOA, CCR3, SRC, PPP1CC, MAPK14, NOX1, RAF1, MAP2K2, MAP2K1, JUN, CCL2, and PRKCA.

  In some embodiments, the target DNA is associated with IL-2 signaling and the DNA is ELK1, MAPK1, PTPN11, AKT2, PIK3CA, SYK, FOS, STAT5B, PIK3CB, PIK3C3, MAPK8, MAPK3 , KRAS, SOCS1, STAT5A, PIK3C2A, LCK, RAF1, MAP2K2, JAK1, AKT1, PIK3R1, MAP2K1, JUN, and AKT3.

  In some embodiments, the target DNA is associated with synaptic long-term suppression, and the DNA is PRKCE, IGF1, PRKCZ, PRDX6, LYN, MAPK1, GNAS, PRKCI, GNAQ, PPP2R1A, IGF1R, PRKD1, MAPK3 , KRAS, GRN, PRKCD, NOS3, NOS2A, PPP2CA, YWHAZ, RAF1, MAP2K2, PPP2R5C, MAP2K1, PRKCA.

  In some embodiments, the target DNA is associated with estrogen receptor signaling, and the DNA is TAF4B, EP300, CARM1, PCAF, MAPK1, NCOR2, SMARCA4, MAPK3, NRIP1, KRAS, SRC, NR3C1 , HDAC3, PPARGC1A, RBM9, NCOA3, RAF1, CREBBP, MAP2K2, NCOA2, MAP2K1, PRKDC, ESR1, and ESR2.

  In some embodiments, the target DNA is associated with a protein ubiquitination pathway, and the DNA is TRAF6, SMURF1, BIRC4, BRCA1, UCHL1, NEDD4, CBL, UBE2I, BTRC, HSPA5, USP7, USP10, It is selected from FBXW7, USP9X, STUB1, USP22, B2M, BIRC2, PARK2, USP8, USP1, VHL, HSP90AA1, and BIRC3.

  In some embodiments, the target DNA is associated with IL-10 signaling and the DNA is TRAF6, CCR1, ELK1, IKBKB, SP1, FOS, NFKB2, MAP3K14, MAPK8, MAPK13, RELA, MAPK14 , TNF, IKBKG, RELB, MAP3K7, JAK1, CHUK, STAT3, NFKB1, JUN, IL1R1, and IL6.

  In some embodiments, the target DNA is associated with VDR / RXR activation and the DNA is PRKCE, EP300, PRKCZ, RXRA, GADD45A, HES1, NCOR2, SP1, PRKC1, CDKN1B, PRKD1, PRKCD , RUNX2, KLF4, YY1, NCOA3, CDKN1A, NCOA2, SPP1, LRP5, CEBPB, FOXO1, and PRKCA.

  In some embodiments, the target DNA is associated with TGF-beta signaling and the DNA is EP300, SMAD2, SMURF1, MAPK1, SMAD3, SMAD1, FOS, MAPK8, MAPK3, KRAS, MAPK9, RUNX2 , SERPINE1, RAF1, MAP3K7, CREBBP, MAP2K2, MAP2K1, TGFBR1, SMAD4, JUN, SMAD5.

  In some embodiments, the target DNA is associated with Toll-like receptor signaling, wherein the DNA is IRAK1, EIF2AK2, MYD88, TRAF6, PPARA, ELK1, IKBKB, FOS, NFKB2, MAP3K14, MAPK8, MAPK13, RELA, TLR4, MAPK14, IKBKG, RELB, MAP3K7, CHUK, NFKB1, TLR2, and JUN are selected.

  In some embodiments, the target DNA is associated with p38 MAPK signaling, the DNA is HSPB1, IRAK1, TRAF6, MAPKAPK2, ELK1, FADD, FAS, CREB1, DDIT3, RPS6KA4, DAXX, MAPK13, TRAF2 , MAPK14, TNF, MAP3K7, TGFBR1, MYC, ATF4, IL1R1, SRF, STAT1.

  In some embodiments, the target DNA is associated with neurotrophin / TRK signaling, and the DNA is NTRK2, MAPK1, PTPN11, PIK3CA, CREB1, FOS, PIK3CB, PIK3C3, MAPK8, MAPK3, KRAS , PIK3C2A, RAF1, MAP2K2, AKT1, PIK3R1, PDPK1, MAP2K1, CDC42, JUN, and ATF4.

  In some embodiments, the target DNA is associated with FXR / RXR activation and the DNA is INS, PPARA, FASN, RXRA, AKT2, SDC1, MAPK8, APOB, MAPK10, PPARG, MTTP, MAPK9 , PPARGC1A, TNF, CREBBP, AKT1, SREBF1, FGFR4, AKT3, and FOXO1.

  In some embodiments, the target DNA is associated with synaptic long-term potentiation, and the DNA is PRKCE, RAP1A, EP300, PRKCZ, MAPK1, CREB1, PRKCI, GNAQ, CAMK2A, PRKD1, MAPK3, KRAS, PRKCD. , PPP1CC, RAF1, CREBBP, MAP2K2, MAP2K1, ATF4, and PRKCA.

  In some embodiments, the target DNA is associated with calcium signaling and the DNA is RAP1A, EP300, HDAC4, MAPK1, HDAC5, CREB1, CAMK2A, MYH9, MAPK3, HDAC2, HDAC7A, HDAC11, HDAC9 , HDAC3, CREBBP, CALR, CAMKK2, ATF4, and HDAC6.

  In some embodiments, the target DNA is associated with EGF signaling and the DNA is ELK1, MAPK1, EGFR, PIK3CA, FOS, PIK3CB, PIK3C3, MAPK8, MAPK3, PIK3C2A, RAF1, JAK1, PIK3R1 , STAT3, MAP2K1, JUN, PRKCA, SRF, and STAT1.

  In some embodiments, the target DNA is associated with hypoxic signaling in the cardiovascular system and the DNA is EDN1, PTEN, EP300, NQO1, UBE2I, CREB1, ARNT, HIF1A, SLC2A4, NOS3, Selected from TP53, LDHA, AKT1, ATM, VEGFA, JUN, ATF4, VHL, and HSP90AA1.

  In some embodiments, the target DNA is associated with LPS / IL-1 mediated inhibition of RXR function, and the DNA is IRAK1, MYD88, TRAF6, PPARA, RXRA, ABCA1, MAPK8, ALDH1A1, GSTP1, It is selected from MAPK9, ABCB1, TRAF2, TLR4, TNF, MAP3K7, NR1H2, SREBF1, JUN, and IL1R1.

  In some embodiments, the target DNA is associated with LXR / RXR activation and the DNA is FASN, RXRA, NCOR2, ABCA1, NFKB2, IRF3, RELA, NOS2A, TLR4, TNF, RELB, LDLR. , NR1H2, NFKB1, SREBF1, IL1R1, CCL2, IL6, and MMP9.

  In some embodiments, the target DNA is associated with amyloid processing, and the DNA is PRKCE, CSNK1E, MAPK1, CAPNS1, AKT2, CAPN2, CAPN1, MAPK3, MAPK13, MAPT, MAPK14, AKT1, PSEN1, It is selected from CSNK1A1, GSK3B, AKT3, and APP.

  In some embodiments, the target DNA is associated with IL-4 signaling and the DNA is AKT2, PIK3CA, PIK3CB, PIK3C3, IRS1, KRAS, SOCS1, PTPN6, NR3C1, PIK3C2A, JAK1, AKT1 , JAK2, PIK3R1, FRAP1, AKT3, and RPS6KB1.

  In some embodiments, the target DNA is associated with cell cycle: G2 / M DNA damage checkpoint regulation, and the DNA is EP300, PCAF, BRCA1, GADD45A, PLK1, BTRC, CHEK1, ATR, CHEK2 , YWHAZ, TP53, CDKN1A, PRKDC, ATM, SFN, and CDKN2A.

  In some embodiments, the target DNA is associated with nitric oxide signaling in the cardiovascular system and the DNA is KDR, FLT1, PGF, AKT2, PIK3CA, PIK3CB, PIK3C3, CAV1, PRKCD, NOS3 , PIK3C2A, AKT1, PIK3R1, VEGFA, AKT3, and HSP90AA1.

  In some embodiments, the target DNA is associated with purine metabolism, and the DNA is NME2, SMARCA4, MYH9, RRM2, ADAR, EIF2AK4, PKM2, ENTPD1, RAD51, RRM2B, TJP2, RAD51C, NT5E, It is selected from POLD1 and NME1.

  In some embodiments, the target DNA is associated with cAMP-mediated signaling, and the DNA is RAP1A, MAPK1, GNAS, CREB1, CAMK2A, MAPK3, SRC, RAF1, MAP2K2, STAT3, MAP2K1, BRAF, And ATF4.

  In some embodiments, the target DNA is associated with mitochondrial dysfunction, and the DNA is selected from SOD2, MAPK8, CASP8, MAPK10, MAPK9, CASP9, PARK7, PSEN1, PARK2, APP, and CASP3 The

  In some embodiments, the target DNA is associated with Notch signaling, and the DNA is selected from HES1, JAG1, NUMB, NOTCH4, ADAM17, NOTCH2, PSEN1, NOTCH3, NOTCH1, and DLL4.

  In some embodiments, the target DNA is associated with the endoplasmic reticulum stress pathway, and the DNA is selected from HSPA5, MAPK8, XBP1, TRAF2, ATF6, CASP9, ATF4, EIF2AK3, and CASP3.

  In some embodiments, the target DNA is associated with pyrimidine metabolism and the DNA is selected from NME2, AICDA, RRM2, EIF2AK4, ENTPD1, RRM2B, NT5E, POLD1, and NME1.

  In some embodiments, the target DNA is associated with Parkinson signaling, and the DNA is selected from UCHL1, MAPK8, MAPK13, MAPK14, CASP9, PARK7, PARK2, and CASP3.

  In some embodiments, the target DNA is associated with cardiac and beta-adrenergic signaling, and the DNA is selected from GNAS, GNAQ, PPP2R1A, GNB2L1, PPP2CA, PPP1CC, and PPP2R5C.

  In some embodiments, the target DNA is associated with glycolysis / gluconeogenesis, and the DNA is selected from HK2, GCK, GPI, ALDH1A1, PKM2, LDHA, and HK1.

  In some embodiments, the target DNA is associated with interferon signaling and the DNA is selected from IRF1, SOCS1, JAK1, JAK2, IFITM1, STAT1, and IFIT3.

  In some embodiments, the target DNA is associated with sonic hedgehog signaling, and the DNA is selected from ARRB2, SMO, GLI2, DYRK1A, GLI1, GSK3B, and DYRKIB.

  In some embodiments, the target DNA is associated with glycerophospholipid metabolism, and the DNA is selected from PLD1, GRN, GPAM, YWHAZ, SPHK1, and SPHK2.

  In some embodiments, the target DNA is associated with phospholipid degradation, and the DNA is selected from PRDX6, PLD1, GRN, YWHAZ, SPHK1, and SPHK2.

  In some embodiments, the target DNA is associated with tryptophan metabolism, and the DNA is selected from SIAH2, PRMT5, NEDD4, ALDH1A1, CYP1B1, and SIAH1.

  In some embodiments, the target DNA is associated with lysine degradation, and the DNA is selected from SUV39H1, EHMT2, NSD1, SETD7, and PPP2R5C.

  In some embodiments, the target DNA is associated with a nucleotide excision repair pathway, and the DNA is selected from ERCC5, ERCC4, XPA, XPC, and ERCC1.

  In some embodiments, the target DNA is associated with starch and sucrose metabolism, and the DNA is selected from UCHL1, HK2, GCK, GPI, and HK1.

  In some embodiments, the target DNA is associated with amino sugar metabolism and the DNA is selected from NQO1, HK2, GCK, and HK1.

  In some embodiments, the target DNA is associated with arachidonic acid metabolism, and the DNA is selected from PRDX6, GRN, YWHAZ, and CYP1B1.

  In some embodiments, the target DNA is associated with circadian rhythm signaling, and the DNA is selected from CSNK1E, CREB1, ATF4, and NR1D1.

  In some embodiments, the target DNA is associated with a coagulation system, and the DNA is selected from BDKRB1, F2R, SERPINE1, and F3.

  In some embodiments, the target DNA is associated with dopamine receptor signaling, and the DNA is selected from PPP2R1A, PPP2CA, PPP1CC, and PPP2R5C.

  In some embodiments, the target DNA is associated with glutathione metabolism and the DNA is selected from IDH2, GSTP1, AMPEP, and IDH1.

  In some embodiments, the target DNA is associated with glycerolipid metabolism and the DNA is selected from ALDH1A1, GPAM, SPHK1, and SPHK2.

  In some embodiments, the target DNA is associated with linoleic acid metabolism and the DNA is selected from PRDX6, GRN, YWHAZ, and CYP1B1.

  In some embodiments, the target DNA is associated with methionine metabolism, and the DNA is selected from DNMT1, DNMT3B, AHCY, and DNMT3A.

  In some embodiments, the target DNA is associated with pyruvate metabolism and the DNA is selected from GLO1, ALDH1A1, PKM2, and LDHA.

  In some embodiments, the target DNA is associated with arginine and proline metabolism, and the DNA is selected from ALDH1A1, NOS3, and NOS2A.

  In some embodiments, the target DNA is associated with eicosanoid signaling and the DNA is selected from PRDX6, GRN, and YWHAZ.

  In some embodiments, the target DNA is associated with fructose and mannose metabolism, and the DNA is selected from HK2, GCK, and HK1.

  In some embodiments, the target DNA is associated with galactose metabolism and the DNA is selected from HK2, GCK, and HK1.

  In some embodiments, the target DNA is associated with stilbene, coumarin, and lignin biosynthesis, and the DNA is selected from PRDX6, PRDX1, and TYR.

  In some embodiments, the target DNA is associated with an antigen presentation pathway, and the DNA is selected from CALR and B2M.

  In some embodiments, the target DNA is associated with steroid biosynthesis, and the DNA is selected from NQO1, and DHCR7.

  In some embodiments, the target DNA is associated with butanoic acid metabolism, and the DNA is selected from ALDH1A1 and NLGN1.

  In some embodiments, the target DNA is associated with a citrate cycle, and the DNA is selected from IDH2 and IDH1.

  In some embodiments, the target DNA is associated with fatty acid metabolism and the DNA is selected from ALDH1A1 and CYP1B1.

  In some embodiments, the target DNA is associated with glycerophospholipid metabolism, and the DNA is selected from PRDX6 and CHKA.

  In some embodiments, the target DNA is associated with histidine metabolism and the DNA is selected from PRMT5 and ALDH1A1.

  In some embodiments, the target DNA is associated with inositol metabolism and the DNA is selected from ERO1L and APEX1.

  In some embodiments, the target DNA is associated with xenobiotic metabolism by cytochrome P450, and the DNA is selected from GSTP1 and CYP1B1.

  In some embodiments, the target DNA is associated with methane metabolism and the DNA is selected from PRDX6 and PRDX1.

  In some embodiments, the target DNA is associated with phenylalanine metabolism, and the DNA is selected from PRDX6 and PRDX1.

  In some embodiments, the target DNA is associated with propanoic acid metabolism, and the DNA is selected from ALDH1A1 and LDHA.

  In some embodiments, the target DNA is associated with selenoamino acid metabolism, and the DNA is selected from PRMT5 and AHCY.

  In some embodiments, the target DNA is associated with sphingolipid metabolism and the DNA is selected from SPHK1 and SPHK2.

  In some embodiments, the target DNA is associated with aminophosphonic acid metabolism, and the DNA is selected from PRMT5.

  In some embodiments, the target DNA is associated with androgen and estrogen metabolism, and the DNA is PRMT5.

  In some embodiments, the target DNA is associated with ascorbic acid and aldaric acid metabolism, and the DNA is ALDH1A1.

  In some embodiments, the target DNA is associated with bile acid biosynthesis and the DNA is ALDH1A1.

  In some embodiments, the target DNA is associated with cysteine metabolism and the DNA is LDHA.

  In some embodiments, the target DNA is associated with fatty acid biosynthesis and the DNA is FASN.

  In some embodiments, the target DNA is associated with glutamate receptor signaling and the DNA is GNB2L1.

  In some embodiments, the target DNA is associated with an NRF2-mediated oxidative stress response and the DNA is PRDX1.

  In some embodiments, the target DNA is associated with the pentose phosphate pathway and the DNA is GPI.

  In some embodiments, the target DNA is associated with pentose and glucuronic acid interconversion, and the DNA is UCHL1.

  In some embodiments, the target DNA is associated with retinol metabolism and the DNA is AFDH1A1.

  In some embodiments, the target DNA is associated with riboflavin metabolism and the DNA is TYR.

  In some embodiments, the target DNA is associated with tyrosine metabolism and the DNA is selected from PRMT5 and TYR.

  In some embodiments, the target DNA is associated with ubiquinone biosynthesis and the DNA is PRMT5.

  In some embodiments, the target DNA is associated with degradation of valine, leucine, and isoleucine, and the DNA is ALDH1A1.

  In some embodiments, the target DNA is associated with lysine degradation and the DNA is ALDH1A1.

  In some embodiments, the target DNA is associated with metabolism of glycine, serine, and threonine, and the DNA is CHKA.

  In some embodiments, the target DNA is associated with pain or taste, and the DNA is TRPM5, TRPA1, TRPM7, TRPC5, TRPC6, TRPC1, Cnrl, cnr2, Grk2, Trpa1, Pomc, Cgrp, Crf , Pka, Era, Nr2b, TRPM5, Prkaca, Prkacb, Prkar1a, and Prkar2a.

  In some embodiments, the target DNA is associated with mitochondrial function, and the DNA is selected from AIF, CytC, SMAC (Diablo), Aifm-1, and Aifm-2.

  In some embodiments, the target DNA is associated with developmental neurology, and the DNA is BMP-4, Chordin (Chrd), Noggin (Nog), WNT (Wnt2, Wnt2b, Wnt3a, Wnt4, Wnt5a , Wnt6, Wnt7b, Wnt8b, Wnt9a, Wnt9b, Wnt10a, Wnt10b, Wnt16), beta-catenin, Dkk-1, Frizzled-related protein, Otx-2, Gbx2, FGF-8, Reelin, Dabl, unc-861 ), Num, Reln.

  Another aspect of the invention is a method for site-specific endonucleolytic cleavage of a single stranded RNA (ssRNA) comprising the step of using a compound, RNP, or composition described herein with a protospacer adjacent motif. (PAM) A method comprising administering in the presence of a presenting oligonucleotide (PAMmer) is provided. Suitable PAMmers suitable for use in the present invention include O'Connell, M. et al. R. et al. Nature, 2014, 516 (7530): 263-6, though not limited thereto.

  Another aspect of the present invention is a method for site-specific endonucleolytic cleavage of single stranded RNA (ssRNA) comprising the CRISPR / Cas type III-B Cmr complex as described herein. There is provided a method comprising administering a compound or RNP comprising, or a composition thereof. In some embodiments, the type III-B Cmr complex may be derived from Pyrococcus furiosus, Sulfolobus solfaricus, or Thermus thermophilus. In some embodiments, the Cmr protein suitable for use in the present invention is Hale, C .; R. et al. Genes & Development, 2014, 28: 2432-2443, and Makarova K. et al. S. et al. Examples include, but are not limited to, those described in Nature Reviews Microbiology, 2015, 13, 1-15.

  Embodiments of the present invention are illustrated by the following examples. However, it is understood that embodiments of the invention are not limited to the specific details of these examples, as those other variations will be known to those skilled in the art or will become apparent in light of this disclosure.

  Unless otherwise specified, starting materials are generally from Aldrich Chemicals Co. (Milwaukee, Wis.), Lancaster Synthesis, Inc. (Wyndham, NH), Acros Organics (Fair Lawn, NJ), Maybridge Chemical Company, Ltd. (Cornwall, England), Tyger Scientific (Princeton, NJ), AstraZeneca Pharmaceuticals (London, England), and Accela ChemBio (San Diego, Calif.).

General Experimental Procedure NMR spectra were recorded for protons at 400 MHz at room temperature on a Varian Unity ™ 400 (available from Varian Inc., Palo Alto, Calif.). Chemical shifts are expressed in parts per million (delta) relative to residual solvent as an internal standard. Peak shapes are shown as follows: s, singlet; d, doublet; dd, doublet doublet; t, triplet; q, quartet; m, multiplet; bs or br . s. , Broad singlet; 2s, two singlets; br. d. Broad doublet. In some cases, only representative ¹ H NMR peaks are shown. Column chromatography is performed in a glass column or Flash 40 Biotage ™ column (ISC, Inc., Shelton, Conn.), Baker ™ silica gel (40 micron, JT Baker, Philipsburg, NJ) or silica gel. 50 (EM Sciences ™, Gibbstown, NJ). MPLC (Medium Pressure Liquid Chromatography) was performed using a Biotage ™ SP purification system from Teledyne ™ Isco ™ or Combiflash ™ Companion ™. Biotage ™ SNAP cartridge KPsil or Redisep Rf silica (from Teledyne ™ Isco ™) was used under low nitrogen pressure. Unless otherwise specified, all reactions were carried out using an anhydrous solvent and under an inert atmosphere of nitrogen gas. Similarly, all reactions were performed at room temperature (about 23 ° C.) unless otherwise specified. When performing TLC (Thin Layer Chromatography), R _f is defined as the ratio of the distance traveled by the compound divided by the distance traveled by the eluent. R _t (retention time). H-Cube® continuous flow hydrogenation reactor: a tabletop independent hydrogenation reactor that combines continuous flow microchemistry with endogenous on-demand hydrogen production and disposable catalyst cartridge systems.

  LC / MS TOF (ESI): All data was collected on an Agilent 1100 LC equipped with an MSD TOF (Agilent model G1969A) mass spectrometric detector operating with an electrospray ionization source. The LC instrument includes a binary pump (Agilent model G1312A) having a pressure upper limit of 400 bar attached to an autosampler (Agilent model G1313A) using external try for sample submission. This column compartment (Agilent model G1316A) is attached to a diode array (Agilent model G1315A). The acquisition of equipment and the handling of data are described in Agilent MassHunter TOF / Q-TOF B.E. 02 (B11285) patch 1.2.3. Elution conditions: Column: No column was used. Flow injection: Injection volume: 1.0 microL, flow rate: 0.5 mL / min. Run time: 1.0 min, solvent: methanol (0.1% formic acid and 0.05% ammonium formate). TOF conditions: ionization source: positive mode electrospray ionization source, gas temperature: 325 ° C., dry gas: 6 L / min, nebulizer: 50 psg, VCap: 3500 V, mass range 110-100 m / z, acquisition rate: 0.99 spectrum / Second: acquisition time, 1012.8 milliseconds / spectrum. All solvents were of HPLC Chromasolv grade from Sigma Aldrich (St. Louis, MO). Most of the chemicals and buffers were purchased from Sigma Aldrich and were all over 97% pure.

  Method C 1.5 min run LRMS (Low Resolution Mass Spectrometry): Waters Acquity HSS T3, 2.1 mm × 50 mm, C18, 1.7 μm, mobile phase: A: 0.1% formic acid aqueous solution (v / v), mobile Phase B: 0.1% formic acid acetonitrile solution (v / v), flow rate -1.25 ml / min, initial conditions: A-95%: B-5%, hold 0.0-0.1 min initially A linear slope is applied from 0.1 to 1.0 minutes to A-5%: B-95%, 1.0-1.1 minutes are held at A-5%: B-95%, 1.1 Return to initial conditions for ~ 1.5 minutes.

Method C 3.0 minute run LRMS (Low Resolution Mass Spectrometry): Waters Acqity
HSS T3, 2.1 mm × 50 mm, C18, 1.7 μm, mobile phase: A: 0.1% formic acid aqueous solution (v / v), mobile phase B: 0.1% formic acid acetonitrile solution (v / v), flow rate -1.25 ml / min, initial conditions: A-95%: B-5%, 0.0-0.1 min hold initially, A-5% over 0.1-2.6 min: B- Applying a linear slope to 95%, 2.6-2.95 minutes hold at A-5%: B-95%, 2.95-3.0 minutes return to initial conditions.

Procedure ((2R, 3S, 4R, 5R, 6R) -5-azido-6-methoxy-3,4-bis ((trimethylsilyl) oxy) tetrahydro-2H-pyran-2-yl) methanol (Ib)

(2R, 3R, 4R, 5R, 6R) -5-azido-2- (hydroxymethyl) -6-methoxytetrahydro-2H-pyran-3,4-diol (Ia) (5 g, 23 mmol) was added to anhydrous pyridine (100 mL) and trimethylsilyl chloride (17.5 mL, 139 mmol) was added. The reaction mixture was stirred at room temperature for 12 hours, after which pyridine was evaporated. The residue was dissolved in ethyl acetate / water. The aqueous phase was extracted once with ethyl acetate and the combined organic layers were washed with water, saturated aqueous sodium chloride solution, dried over magnesium sulfate, filtered and concentrated to a corresponding 9.9 g (98% yield). The persilylated compound was obtained as a yellow oil. This material was used in the next step without further purification. To a solution of the above persilylated compound (9.71 g, 22.3 mmol) in anhydrous methanol (45 mL) cooled to 0 ° C., 9.06 mL of a potassium carbonate methanol solution (0.032 M) was added. The reaction mixture was stirred at 0 ° C. for 1 hour and then neutralized by the addition of 17 microL acetic acid. The solvent was evaporated and the residue was dissolved in ethyl acetate. Water was added and the aqueous phase was extracted twice with ethyl acetate. The combined organic layers were washed with brine, dried over magnesium sulfate, filtered and concentrated. The crude material was purified through silica gel by flash chromatography (30% ethyl acetate / hexanes) to give 6.77 g (84%) of (Ib) as an oil. [Α] _D 7 (c 1, chloroform); ¹ H NMR (400 MHz, chloroform-d) delta ppm 0.14 (s, 9H), 0.20 (s, 9H), 1.80 (br. S) , 1H), 3.36-3.42 (m, 1H), 3.45 (dd, J = 7.3, 4.6 Hz, 1H), 3.54 (dd, J = 10.0, 8.0 Hz, 1H), 3.59 (s, 3H), 3.65 (dd, J = 11.3, 4.7 Hz, 1H), 3.77 (d, J = 2.7 Hz, 1H), 3.87 (dd, J = 11.2, 7.3 Hz, 1H), 4.14 (d, J = 8.0 Hz, 1H); ¹³ C NMR (100 MHz, chloroform-d) Delta ppm 0.27 (3C), 0.6 (3C), 57.3, 62.6, 64.0, 71 1, 73.7, 75.2, 103.4; HRMS (ESI) C 13 H 29 N 3 O 5 Si 2 (m / z) [M + Na] calcd for ⁺ 386.1538, found 386. 1539.

(3R, 4R, 5R, 6R) -5-azido-2,2-bis (hydroxymethyl) -6-methoxytetrahydro-2H-pyran-3,4-diol (Ic)
(Ib) (7.73 g, 21.3 mmol) was dissolved in dichloromethane (70 mL). Dimethyl sulfoxide (10.6 mL, 150 mmol) and triethylamine (9 mL, 60 mmol) were added and the reaction mixture was cooled to 0 ° C. Sulfur trioxide pyridine complex (10.2 g, 64 mmol) was added and the mixture was stirred at 0 ° C. for 1 hour and then allowed to warm to room temperature over 30 minutes. The reaction was quenched with saturated sodium chloride solution and diluted with dichloromethane. The aqueous phase was extracted 3 times with dichloromethane and the combined organic layers were washed with saturated aqueous sodium chloride solution, dried over magnesium sulfate, filtered and concentrated to give the corresponding aldehyde. The aldehyde was dissolved in absolute ethanol (106 mL) and paraformaldehyde powder (40.3 g, 425 mmol) was added followed by a 21 wt% ethanol solution of sodium ethoxide (16 mL, 42.5 mmol). The reaction mixture was stirred at room temperature for 12 hours, after which the ethanol was evaporated. Methanol was added to the crude mixture and the solid was filtered and rinsed thoroughly with methanol. Silica gel was added to the filtrate containing the desired product and the methanol was evaporated. The resulting dry load was dried under high vacuum and loaded onto the column. The crude material was purified through silica gel by flash chromatography (10% methanol / dichloromethane) to give 3.03 g of (Ic) as a colorless oil (57% over 2 steps). [Α] _D −20 (c 1.25, methanol); ¹ H NMR (400 MHz, methanol-d ₄ ) delta ppm 3.46 (dd, J = 10.2, 8.1 Hz, 1H), 3 .51 (s, 3H), 3.64-3.80 (m, 5H), 3.80-3.83 (m, 1H), 4.54 (d, J = 8.0 Hz, 1 H) ¹³ C NMR (100 MHz, methanol-d ₄ ) delta ppm 57.2, 61.2, 63.6, 65.9, 69.9, 71.2, 80.9, 101.2; HRMS (ESI ) Calculated value for C ₈ H ₁₅ N ₃ O ₆ (m / z) [M + Na] ⁺ 272.0853, found value 272.0856.

N-((3aR, 4S, 7S, 8R, 8aR) -4- (hydroxymethyl) -2,2-dimethylhexahydro-4,7-epoxy [1,3] dioxolo [4,5-d] oxepin- 8-yl) acetamide (Ie-1)
A solution of compound (3) (230 mg, 0.986 mmol) in 6.6 mL of dimethylformamide was added to 2,2-dimethoxypropane (0.8 mL, 6 mmol) followed by (+/−)-camphor-10-sulfonic acid ( 101 mg, 0.435 mmol) was added. The reaction mixture was stirred at 70 ° C. for 24 hours, cooled to room temperature, and then methanol was added (1.2 mL). The reaction mixture was stirred at room temperature for 30 minutes and then neutralized with triethylamine (56 microL). The solvent was evaporated and the residue was coevaporated 3 times with toluene. The crude material was purified through silica gel by flash chromatography (15/1 ethyl acetate / methanol) to give compound (Ie-1) as a white solid (246 mg, 91% yield). m. p. [Α] _D 147 (c 1, methanol); ¹ H NMR (400 MHz, methanol-d ₄ ) delta ppm 1.34 (s, 3H), 1.48 (s): 164.7-166.0 ° C. 3H), 1.98 (s, 3H), 3.77 (d, J = 7.8 Hz, 1H), 3.83 (d, J = 7.8 Hz, 1H), 3.86 (d, .J = 11.6 Hz, 1H), 3.90 (d, .J = 11.3 Hz, 1H), 3.91-3.94 (m, 1H), 4.14-4.19 (m , 1H), 4.29 (d, J = 6.0 Hz, 1H), 5.23 (d, J = 2.0 Hz, 1H); ¹³ C NMR (100 MHz, methanol-d ₄ ) delta ppm 22.7, 26.9, 28.5, 56.8, 61.9, 70.2, 76.1, 76. , 83.0, 102.6, 112.5, 173.6 ; HRMS (ESI) C 12 H 19 NO 6 (m / z) [M + H] calcd for ⁺ 274.1285, found 274.1274 .

(1S, 2R, 3R, 4R, 5S) -4-Azido-1- (hydroxymethyl) -6,8-dioxabicyclo [3.2.1] octane-2,3-diol (1)
Tetraol (Ic) (3 g, 12 mmol) was dissolved in water (40 mL) and concentrated sulfuric acid (6.7 mL) was added. The reaction mixture was stirred at 100 ° C. for 40 hours, cooled to room temperature, and then neutralized by the addition of concentrated ammonium hydroxide. Water was evaporated and methanol was added to the resulting mixture. The solid was filtered and rinsed thoroughly with methanol. Silica gel was added to the filtrate containing the desired product and the methanol was evaporated. The resulting dry load was dried under high vacuum and loaded onto the column. The crude material was purified through silica gel by flash chromatography (10% methanol / dichloromethane) to give 2.2 g (84%) of (1) as a colorless oil. [Α] _D 160 (c 1.1, methanol); ¹ H NMR (400 MHz, methanol-d ₄ ) delta ppm 3.35 (dd, J = 9.2, 1.6 Hz, 1H), 70 (d, J = 8.2 Hz, 1H), 3.76 (d, J = 8.0 Hz, 1H), 3.80 (d, .J = 11.3 Hz, 1H), 3.83 -3.89 (m, 2H), 3.90 (d, J = 11.5 Hz, 1H), 5.32 (d, J = 1.4 Hz, 1H); ¹³ C NMR (100 MHz, methanol) -d ₄₎ delta ppm 61.9, 66.1, 69.5, 69.6 , 71.0, 85.3, 102.7; HRMS (ESI) C 7 H 11 N 3 O 5 (m / z ) Calculated value 240.0591 for [M + Na] ⁺ , measured value 240.0596.

(1R, 2R, 3R, 4R, 5S) -4-acetamido-1- (acetoxymethyl) -6,8-dioxabicyclo [3.2.1] octane-2,3-diyldiacetate (2)
In a round bottom flask, compound (1) (1.93 g, 8.9 mmol) was dissolved in ethanol (45 mL) and the system was flushed with nitrogen. Lindlar catalyst (1.89 g, 0.9 mmol) was added and the system was flushed with nitrogen and then with hydrogen. The reaction mixture was stirred at room temperature under a hydrogen atmosphere (using a balloon) for 24 hours. The palladium was filtered through a nylon membrane and rinsed thoroughly with methanol and then water. The solvent was evaporated and the residue was dissolved in water and lyophilized. The resulting crude material was then dissolved in pyridine (40 mL) and acetic anhydride was added (9 mL, 100 mmol). The reaction mixture was stirred at room temperature for 48 hours and the pyridine was evaporated. The residue was dissolved in ethyl acetate and washed with saturated sodium bicarbonate solution. The aqueous phase was extracted twice with ethyl acetate, after which the combined organic layers were washed with saturated aqueous sodium chloride solution, dried over magnesium sulfate, filtered and evaporated. The crude material was purified through silica gel by flash chromatography (3% methanol / dichloromethane) to give (2) (3.19 g, quantitative). [Α] _D 75 (c 1, chloroform); ¹ H NMR (400 MHz, methanol-d ₄ ) delta ppm 1.95 (s, 3H), 1.95 (s, 3H), 2.04 (s, 3H), 2.15 (s, 3H), 3.75 (d, J = 8.6 Hz, 1H), 4.06 (d, J = 8.6 Hz, 1H), 4.13 (d, J = 11.6 Hz, 1H), 4.20 (d, J = 10.6 Hz, 1H), 4.46 (d, J = 11.3 Hz, 1H), 5.13 (dd, J = 10.4, 4.4 Hz, 1H), 5.35 (d, J = 1.0 Hz, 1H), 5.38 (d, J = 4.3 Hz, 1H); ¹³ C NMR (100 MHz , methanol -d ₄₎ delta ppm 20.6, 20.7 (2C), 22.6, 53.3, 63.0, 68.9, 69.1, 70.3, 82.6, 103.0, 171.8, 171.9, 172.1, 173.8; HRMS (ESI) C ₁₅ H ₂₁ NO ₉ (m / z ) Calculated value 360.1289 for [M + H] ⁺ , measured value 360.1290.

N-((1S, 2R, 3R, 4R, 5S) -2,3-dihydroxy-1- (hydroxymethyl) -6,8-dioxabicyclo [3.2.1] octane-4-yl) acetamide ( 3)
Compound (2) (3.19 g, 8.88 mmol) was dissolved in tetrahydrofuran (50 mL) and 0.5 M sodium methoxide in methanol (100 mL, 50 mmol) was added. The reaction mixture was stirred for 12 hours at room temperature and then neutralized by the addition of H ⁺ Amberlyte ™ IR-120 resin. The resin was filtered and the solvent evaporated to give 1.71 g of (3) as a white solid (83%). m. p. : 175.7-176.1 ° C; [α] _D 164 (c 1, methanol); ¹ H NMR (400 MHz, methanol-d ₄ ) delta ppm 1.99 (s, 3H), 3.68 (d , J = 8.1 Hz, 1H), 3.70-3.73 (m, 1H), 3.75 (d, J = 7.8 Hz, 1H), 3.81 (d, J = 11.3) Hz, 1H), 3.87 (d, J = 4.3 Hz, 1H), 3.92 (d, J = 11.3 Hz, 1H), 3.95 (dd, J = 9.9, 1 .1 Hz, 1 H), 5.22 (d, J = 1.3 Hz, 1 H); ¹³ C NMR (100 MHz, methanol-d ₄ ) delta ppm 22.7, 56.4, 62.1, 69.2, 69.3, 70.6, 85.1, 102.8, 174.1; HRMS (ES _{) C 9 H 15 NO 6 (} m / z) [M + H] calcd for ⁺ 234.0972, found 234.0974.

Benzyl (4-((2-((1- (1-((1S, 2R, 3R, 4R, 5S) -4-acetamido-2,3-dihydroxy-6,8-dioxabicyclo [3.2. 1] Octane-1-yl) -2,5,8,11-tetraoxatridecan-13-yl) -1H-1,2,3-triazol-4-yl) methoxy) ethyl) amino) -4- Oxobutyl) carbamate (4), benzyl (4-((1,3-bis ((1- (1-((1S, 2R, 3R, 4R, 5S) -4-acetamido-2,3-dihydroxy-6, 8-dioxabicyclo [3.2.1] octane-1-yl) -2,5,8,11-tetraoxatridecan-13-yl) -1H-1,2,3-triazol-4-yl ) Methoxy) propan-2-yl) amino) -4-oxobut ) Carbamate (5), benzyl (4-((1,3-bis ((1- (1-((1S, 2R, 3R, 4R, 5S) -4-acetamido-2,3-dihydroxy-6, 8-dioxabicyclo [3.2.1] octane-1-yl) -2,5,8,11-tetraoxatridecan-13-yl) -1H-1,2,3-triazol-4-yl ) Methoxy) -2-(((1- (1-((1S, 2R, 3R, 4R, 5S) -4-acetamido-2,3-dihydroxy-6,8-dioxabicyclo [3.2.1 ] Octan-1-yl) -2,5,8,11-tetraoxatridecan-13-yl) -1H-1,2,3-triazol-4-yl) methoxy) methyl) propan-2-yl) Amino) -4-oxobutyl) carbamate (6)

In a microwave vial, compound (Ie-1) (50 mg, 0.18 mmol) was dissolved in 1 mL of dichloromethane. 12.5 M aqueous sodium hydroxide (0.5 mL), followed by 15-crown-5-ether (5 microL, 0.02 mmol) and 1-azido-2- (2- (2- (2-iodoethoxy) ) Ethoxy) ethoxy) ethane (described in J. Am. Chem. Soc. 132, 1523 (2010)) (301 mg, 0.915 mmol) was added. The reaction mixture was stirred vigorously at 55 ° C. for 24 hours. The organic phase was removed, dried over magnesium sulfate, filtered and concentrated. The crude material was purified through silica gel by flash chromatography (5% methanol / ethyl acetate) to give compound (Ie-2) as an oil (52 mg, 60% yield). [Α] _D 74 (c 1, chloroform); ¹ H NMR (400 MHz, methanol-d ₄ ) delta ppm 1.34 (s, 3H), 1.49 (s, 3H), 1.98 (s, 3H), 3.37 (t, J = 4.9 Hz, 2H), 3.62-3.71 (m, 14H), 3.75-3.80 (m, 2H), 3.86 (d , J = 8.1 Hz, 1H) 3.90-3.97 (m, 2H), 4.12-4.19 (m, 1H), 4.31 (d, J = 5.8 Hz, 1H) , 5.23 (d, J = 2.0 Hz, 1H); ¹³ C NMR (100 MHz, methanol-d ₄ ) delta ppm 22.7, 26.9, 28.5, 51.9, 56.7, 70.9, 71.1, 71.3, 71.6, 71.7, 71.8, 71.81, 71 .82, 72.7, 76.1, 76.5, 82.1, 102.4, 112.4, 173.6; HRMS (ESI) C ₂₀ H ₃₄ N ₄ O ₉ (m / z) [M + H] ⁺ Calculated value 475.2399, Actual value 475.2386.

The intermediate (If-1) is known and is described in WO06120545.
Intermediate (If-2) can be synthesized as follows: Tetrabutylammonium iodide (287 mg, 0 mg) in a solution of Boc-serinol (1000 mg, 5.1 mmol) in tetrahydrofuran (21 mL) at room temperature. .76 mmol), sodium iodide (153 mg, 1.02 mmol), and propargyl bromide (1.8 mL, 16 mmol, 80% toluene solution) were added. Potassium hydroxide (569 mg, 10.1 mmol) was added in small portions over 30 minutes, after which the mixture was stirred at room temperature for 16 hours. The reaction mixture was diluted with ethyl acetate and water. The aqueous phase was extracted once with ethyl acetate. The combined organic layers were dried over magnesium sulfate, filtered and concentrated. The crude material was purified by flash chromatography (30% ethyl acetate / hexane) through silica gel to give compound (If-2) as an oil (530 mg, 39% yield). ¹ H NMR (400 MHz, chloroform-d / TMS) delta ppm 1.44 (s, 9H), 2.44 (t, J = 2.4 Hz, 2H), 3.53-3.67 (m, 4H), 3.92 (br.s., 1H), 4.16 (d, J = 2.5 Hz, 4H), 4.90 (br.s., 1H); ¹³ C NMR (100 MHz, Chloroform-d / TMS) Delta ppm 28.4 (3C), 49.5, 58.5 (2C), 68.6 (2C), 74.6 (2C), 77.2, 79.5 (2C) HRMS (ESI) C ₁₄ H ₂₁ NO ₄ (m / z) [M + H] ⁺ Calculated 268.1543, Found 268.1536.

Intermediate (If-3) is known and is described in R.I. Roy et al. J. et al. Org. Chem. 73, 5602 (2008).
The compound (If-1), (If-2), or (If-3) (1 equivalent) is dissolved in dichloromethane (0.2M), 4M hydrogen chloride in dioxane (5-5). 10 equivalents) was added. The reaction mixture was stirred at room temperature for 2-3 hours, after which the solvent was evaporated. The residue was dried under high vacuum for 1 hour. The resulting intermediate was used in the next step without further purification. The intermediate obtained above (1 eq) and 4-(((benzyloxy) carbonyl) amino) butanoic acid (1 eq) were dissolved in a mixture of dioxane and dimethylformamide (0.09M, 3: 1). . (Benzotriazol-1-yloxy) tripyrrolidinophosphonium hexafluorophosphate (1.2 eq) was added followed by N, N-diisopropylethylamine (5 eq). The reaction mixture was stirred at room temperature for 16 hours. Dichloromethane and water were added and the aqueous phase was extracted twice with dichloromethane. The combined organic layers were dried over magnesium sulfate, filtered and concentrated. This crude material was dissolved in a minimum amount of toluene, loaded onto a column and purified by flash chromatography through silica gel.

Intermediate (I-g-1): Purification conditions: 100% ethyl acetate, quantitative, oil. ¹ H NMR (400 MHz, chloroform-d / TMS) delta ppm 1.80-1.91 (m, 2H), 2.24 (t, J = 7.1 Hz, 2H), 2.46 (t, J = 2.3 Hz, 1H), 3.22-3.31 (m, 2H), 3.43-3.51 (m, 2H), 3.56-3.64 (m, 2H), 4 .16 (d, J = 2.3 Hz, 2H), 5.07 (br.s., 1H), 5.10 (s, 2H), 6.09 (br.s., 1H), 7. 28-7.42 (m, 5H); ¹³ C NMR (100 MHz, chloroform-d / TMS) delta ppm 25.9, 33.7, 39.1, 40.5, 58.3, 66.7, 68.7, 74.8, 79.4, 128.1, 128.5 (4C), 136.6 156.7, 172.5; HRMS (ESI) C 17 H 22 N 2 O 4 (m / z) [M + H] calcd for ⁺ 319.1652, found 319.1646.

Intermediate (Ig-2): Purification conditions: 70% ethyl acetate / hexane, 65 mg, oil (76% yield), oil. ¹ H NMR (400 MHz, chloroform-d / TMS) delta ppm 1.79-1.91 (m, 2H), 2.24 (t, J = 7.1 Hz, 2H), 2.44 (t, J = 2.4 Hz, 2H), 3.20-3.29 (m, 2H), 3.54-3.69 (m, 4H), 4.16 (d, J = 1.5 Hz, 4H ), 4.22-4.33 (m, 1H), 5.10 (br.s, 3H), 6.04 (d, J = 7.8 Hz, 1H), 7.28-7.42 ( m, 5H); ¹³ C NMR (100 MHz, chloroform-d / TMS) delta ppm 25.8, 33.7, 40.4, 48.2, 58.4 (2C), 66.6, 68.3 (2C), 74.7 (2C), 79.4 (2C), 128.1, 128.5 ( C), 136.6, 156.6, 172.2 ; HRMS (ESI) C 21 H 26 N 2 O 5 (m / z) [M + H] calcd for ⁺ 387.1914, found 387.1904 .

Intermediate (I-g-3): purification conditions: 70% ethyl acetate / hexane, 42 mg, oil (yield 60%). ¹ H NMR (400 MHz, chloroform-d) delta ppm 1.76-1.88 (m, 2H), 2.21 (t, J = 7.1 Hz, 2H), 2.44 (t, J = 2.3 Hz, 3H), 3.18-3.30 (m, 2H), 3.84 (s, 6H), 4.14 (d, J = 2.3 Hz, 6H), 5.10 ( s, 2H), 5.12 (br. s., 1H), 5.89 (br. s., 1H), 7.28-7.40 (m, 5H); ¹³ C NMR (100 MHz, chloroform -D / TMS) Delta ppm 25.7, 34.3, 40.3, 58.6 (3C), 59.2, 66.6, 68.5 (3C), 74.6 (3C), 79. 5 (3C), 128.1, 128.5 (4C), 136.6, 156.6, 17 _{.6; HRMS (ESI) C 25} H 30 N 2 O 6 (m / z) [M + H] calcd for ⁺ 455.2177, found 455.2167.

Intermediate (Ih-1):
Tris (3-hydroxypropyltriazolylmethyl) amine (THPTA; see MG Finn et al. In Agewandte Chemie International Edition 48, 9879 (2009)) (2 mg, 0.005 mmol) and copper sulfate (1 mg, 0 .004 mmol) in water (50 microL), then (Ie-2) (42 mg, 0.089 mmol) and alkyne (Ig-1) (40 mg, 0.125 mmol) in methanol (0. 9 mL) was added to the solution. Then sodium ascorbate (1.8 mg, 0.009 mmol) dissolved in water (30 microL) was added and the reaction mixture was stirred at room temperature for 24 hours. The solvent was evaporated and the crude material was purified through silica gel by flash chromatography (5-10% methanol in dichloromethane) to give the desired compound (Ih-1) as an oil (54 mg, yield 76). %). [Α] _D 48.2 (c 0.54, methanol); ¹ H NMR (400 MHz, methanol-d ₄ ) delta ppm 1.33 (s, 3H), 1.48 (s, 3H), 70-1.83 (m, 2H), 1.98 (s, 3H), 2.21 (t, J = 7.4 Hz, 2H), 3.13 (t, J = 6.9 Hz, 2H) ), 3.37 (t, J = 5.4 Hz, 2H), 3.51-3.70 (m, 14H), 3.71-3.95 (m, 7H), 4.15 (t, J = 6.5 Hz, 1H), 4.29 (d, J = 5.8 Hz, 1H), 4.56 (t, J = 5.0 Hz, 2H), 4.60 (s, 2H) , 5.06 (s, 2H), 5.22 (d, J = 1.8 Hz, 1H), 7.25-7.38 (m, 5H), 8.0 1 (s, 1 H); ¹³ C NMR (100 MHz, methanol-d ₄ ) delta ppm 22.7, 27.0, 27.4, 28.5, 34.4, 40.5, 41.4, 51.6, 56.7, 62.4, 64.9, 67.5, 70.0, 70.5, 70.8, 71.1, 71.5, 71.6, 71.65, 71. 7, 71.73, 72.6, 73.8, 76.2, 76.5, 82.1, 102.4, 112.4, 126.1, 129.0, 129.1, 129.6, 138.6, 146.1, 159.0, 173.6, 175.7; HRMS (ESI) calculated for C ₃₇ H ₅₆ N ₆ O ₁₃ (m / z) [M + H] ⁺ 793.3978, Actual value 793.3959.

Intermediate (Ih-2)
THPTA (22 mg, 0.051 mmol) and copper sulfate (2.5 mg, 0.01 mmol) were dissolved in water (70 microL), then (Ie-2) (48 mg, 0.1 mmol) and alkyne (I -G-2) (20 mg, 0.051 mmol) was added to a solution of methanol (1 mL). Then sodium ascorbate (4 mg, 0.02 mmol) dissolved in water (30 microL) was added and the reaction mixture was stirred at room temperature for 72 hours. The solvent was evaporated and the residue was dissolved in dichloromethane and saturated aqueous ammonium chloride. The aqueous phase was extracted 3 times with dichloromethane. The combined organic layers were dried over magnesium sulfate, filtered and concentrated. This crude material was used in the next step without further purification.

Intermediate (Ih-3)
THPTA (34 mg, 0.079 mmol) and copper sulfate (4 mg, 0.016 mmol) were dissolved in water (200 microL), then (Ie-2) (50 mg, 0.1 mmol) and alkyne (I-g -3) (24 mg, 0.053 mmol) was added to a methanol (1 mL) solution. Then sodium ascorbate (6.5 mg, 0.032 mmol) dissolved in water (30 microL) was added and the reaction mixture was stirred at room temperature for 72 hours. The solvent was evaporated and the residue was dissolved in dichloromethane and saturated aqueous ammonium chloride. The aqueous phase was extracted 3 times with dichloromethane. The combined organic layers were dried over magnesium sulfate, filtered and concentrated. This crude material was used in the next step without further purification.

General Procedure for Acetonide Removal Compound (Ih-1), (Ih-2), or (Ih-3) (0.030-0.068 mmol) is mixed with acetic acid, methanol, and water. (1.6-1.8 mL, 0.5 mL, and 0.5 mL, respectively) and stirred at 70 ° C. for 24 hours. The solvent was evaporated and the residue was coevaporated twice with toluene. The crude material was purified through silica gel by flash chromatography.

(4):
Purification conditions: 10% methanol in dichloromethane, 43.3 mg, oil (yield 85%). [Α] _D 45 (c 1, methanol); ¹ H NMR (400 MHz, methanol-d ₄ ) delta ppm 1.72-1.83 (m, 2H), 1.99 (s, 3H), 22 (t, J = 7.4 Hz, 2H), 3.13 (t, J = 6.8 Hz, 2H), 3.37 (t, J = 5.4 Hz, 2H), 3.52 − 3.79 (m, 20H), 3.85-4.00 (m, 3H), 4.57 (t, J = 5.0 Hz, 2H), 4.61 (s, 2H), 5.07 (s, 2H), 5.21 ( s, 1H), 7.24 - 7.41 (m, 5H), 8.02 (s, 1H); 13 C NMR (100 MHz, methanol -d ₄₎ delta ppm 22.8, 27.4, 34.4, 40.4, 41.4, 51.6, 56.4, 64. 9, 67.5, 69.0, 70.0, 70.1, 70.4, 70.5, 71.4, 71.5 (2C), 71.6, 71.65, 71.7, 72 .5, 84.3, 102.6, 126.0, 129.0 (2C), 129.1, 129.6 (2C), 138.6, 145.8, 159.0, 174.0, 175 HRMS (ESI) calculated for C ₃₄ H ₅₂ N ₆ O ₁₃ (m / z) [M + H] ⁺ 753.3665, found 753.3679.

(5):
Purification conditions: 20% methanol in dichloromethane, 25 mg, oil (20% yield over two steps). [Α] _D 56 (c 1.25, methanol); ¹ H NMR (400 MHz, methanol-d ₄ ) delta ppm 1.72-1.81 (m, 2H), 1.99 (s, 6H), 2.23 (t, J = 7.5 Hz, 2H), 3.13 (t, J = 6.9 Hz, 2H), 3.50-3.80 (m, 36H), 3.85-3 .91 (m, 6H), 3.92-4.00 (m, 4H), 4.13-4.25 (m, 1H), 4.52-4.63 (m, 8H), 5.07 (S, 2H), 5.21 (d, J = 1.3 Hz, 2H), 7.23-7.40 (m, 5H), 8.01 (s, 2H); ¹³ C NMR (100 MHz , methanol -d ₄₎ delta ppm 22.6 (2C), 27.2, 34.2, 41.2, 50. 2, 51.4 (2C), 56.2 (2C), 65.0 (2C), 67.3, 68.8 (2C), 69.9 (2C), 70.0 (2C), 70. 2 (2C), 70.3 (2C), 71.2 (2C), 71.3 (4C), 71.4 (2C), 71.5 (2C), 71.52 (2C), 72.3 (2C), 84.1 (2C), 102.4 (2C), 125.8 (2C), 128.8 (2C), 128.9, 129.4 (2C), 138.4, 145.6 (2C), 158.8, 173.8 ( 2C), 175.3; HRMS (ESI) C 55 H 86 N 10 O 23 (m / z) [M + H] calcd for ⁺ 1255.5940, Found Value 12555.5925.

(6):
Purification conditions: 20% methanol in dichloromethane, 31 mg, oil (18% yield over two steps). [Α] _D 53 (c 1, methanol); ¹ H NMR (400 MHz, methanol-d ₄ ) delta ppm 1.65-1.78 (m, 2H), 1.98 (s, 9H), 19 (t, J = 7.3 Hz, 2H), 3.11 (t, J = 6.8 Hz, 2H), 3.51-3.80 (m, 54H), 3.86-3.91 (M, 9H), 3.91-3.99 (m, 6H), 4.51-4.63 (m, 12H), 5.06 (s, 2H), 5.21 (d, J = 1 .3 Hz, 3H), 7.24 - 7.40 (m, 5H), 7.98 (s, 3H); HRMS (ESI) C 76 H 120 N 14 O 33 (m / z) [M + 2H ] Calculated value 8799.4144 for ⁺ / 2, measured value 8799.4148.

N- (2-((1- (1-((1S, 2R, 3R, 4R, 5S) -4-acetamido-2,3-dihydroxy-6,8-dioxabicyclo [3.2.1] octane -1-yl) -2,5,8,11-tetraoxatridecan-13-yl) -1H-1,2,3-triazol-4-yl) methoxy) ethyl) -4-aminobutanamide (7 ), 4-amino-N- {1,3-bis [(1- {1-[(1S, 2R, 3R, 4R, 5S) -4- (acetylamino) -2,3-dihydroxy-6,8 -Dioxabicyclo [3.2.1] oct-1-yl] -2,5,8,11-tetraoxatridecan-13-yl} -1H-1,2,3-triazol-4-yl) Methoxy] propan-2-yl} butanamide (8), 4-amino-N- (1,3- [[1- {1-[(1S, 2R, 3R, 4R, 5S) -4- (acetylamino) -2,3-dihydroxy-6,8-dioxabicyclo [3.2.1] octa- 1-yl] -2,5,8,11-tetraoxatridecan-13-yl} -1H-1,2,3-triazol-4-yl) methoxy] -2-{[(1- {1- [(1S, 2R, 3R, 4R, 5S) -4- (acetylamino) -2,3-dihydroxy-6,8-dioxabicyclo [3.2.1] oct-1-yl] -2,5 , 8,11-Tetraoxatridecan-13-yl} -1H-1,2,3-triazol-4-yl) methoxy] methyl} propan-2-yl) butanamide (9)

In a round bottom flask, compound (4), (5), or (6) (1 eq) was dissolved in methanol (0.01 M) and the flask was flushed with nitrogen. Palladium on carbon (10%, 0.7 eq) was added and the flask was flushed with nitrogen and then with hydrogen. The reaction mixture was stirred at room temperature for 12-24 hours under a hydrogen atmosphere (using a hydrogen filled balloon). The palladium was filtered using 0.45 microm PTFE Acrodisc Cr and rinsed once with methanol. The solvent was evaporated.

(7):
25.5 mg, oil, 76% yield; [α] _D 57.6 (c 1.25, methanol); ¹ H NMR (400 MHz, methanol-d ₄ ) delta ppm 1.70-1.81 (m , 2H), 1.99 (s, 3H), 2.24 (t, J = 7.4 Hz, 2H), 2.67 (t, J = 6.8 Hz, 2H), 3.36-3 .41 (m, 2H), 3.51-3.80 (m, 19H), 3.84-4.01 (m, 4H), 4.59 (t, J = 5.2 Hz, 2H), 4.61 (s, 2H), 5.21 (s, 1H), 8.03 (s, 1H); ¹³ C NMR (100 MHz, methanol-d ₄ ) delta ppm 22.8, 29.6, 34 .5, 40.4, 42.0, 51.6, 56.4, 64.9, 69.0, 70.0 70.1, 70.5, 70.6, 71.4, 71.5 (2C), 71.6, 71.66, 71.7, 72.5, 84.3, 102.6, 126.0 , 145.8, 174.1, 175.9; HRMS (ESI) calculated for C ₂₆ H ₄₆ N ₆ O ₁₁ (m / z) [M + H] ⁺ 619.3297, found 619.3278.

(8):
This crude material was dissolved in 0.5 mL methanol / water (50:50) and injected onto an HPLC column. Preparative high performance liquid chromatography (HPLC) uses a Waters XBridge BEH C18 OBD preparative column, 130Å, 5 μm, 19 mm × 100 mm (Waters, part no. 18620278), eluting with a linear gradient at a flow rate of 17 mL / min. I went. Solvent gradient: acetonitrile / water / trifluoroacetic acid (2: 98: 0.1) to (22: 58: 0.1) over 40 minutes. Collected fractions were analyzed by analytical LCMS and fractions 25.7-27.3 min determined to be of sufficient purity were pooled and evaporated to give 10.7 mg of (8) as an oil. It was. Yield 49%; [α] _D 56 (c 1, methanol); ¹ H NMR (500 MHz, methanol-d ₄ ) delta ppm 1.86-1.95 (m, 2H), 1.99 (s, 6H), 2.37 (t, J = 7.0 Hz, 2H), 2.96 (t, J = 7.4 Hz, 2H), 3.50-3.80 (m, 36H), 3. 84-4.00 (m, 10H), 4.17-4.26 (m, 1H), 4.57-4.62 (m, 8H), 5.21 (s, 2H), 8.03 ( s, 2 H); ¹³ C NMR (100 MHz, methanol-d ₄ ) delta ppm 22.8 (2C), 29.2, 34.5, 41.8, 50.4, 51.6 (2C), 56.4 (2C), 65.1 (2C), 69.0 (2C), 70.1 (2C), 7 .3 (2C), 70.5 (2C), 70.6 (2C), 71.4 (2C), 71.5 (4C), 71.6 (2C), 7.67, (2C), 71 7 (2C), 72.5 (2C), 84.3 (2C), 102.6 (2C), 126.1 (2C), 145.8 (2C), 174.1 (2C), 175. 6; HRMS (ESI) C ₄₇ H ₈₀ N ₁₀ O ₂₁ (m / z) Calculated for [M + H] ⁺ 1121.5572, found 1121.5558.

(9):
The crude material was dissolved in 0.5 mL methanol / water (50:50) and injected onto an HPLC column. Preparative high performance liquid chromatography (HPLC) uses a Waters XBridge BEH C18 OBD preparative column, 130Å, 5 μm, 19 mm × 100 mm (Waters, part no. 18620278), eluting with a linear gradient at a flow rate of 17 mL / min. I went. Solvent gradient: acetonitrile / water / trifluoroacetic acid (2: 98: 0.1) to (22: 58: 0.1) over 40 minutes. The collected fractions were analyzed by analytical LCMS and fractions of 30.3-32.0 minutes that were determined to be of sufficient purity were pooled and evaporated to give 15 mg of (9) as an oil. Yield 63%; [α] _D 59.1 (c 1.1, methanol); ¹ H NMR (500 MHz, methanol-d ₄ ) delta ppm 1.84-1.92 (m, 2H), 00 (s, 9H), 2.31-2.38 (m, 2H), 2.97 (t, J = 7.3 Hz, 2H), 3.54-3.80 (m, 54H), 3 .86-3.93 (m, 9H), 3.93-4.00 (m, 6H), 4.57 (s, 6H), 4.60 (t, J = 4.9 Hz, 6H), 5.22 (s, 3H), 8.02 (s, 3H); HRMS (ESI) C 68 H 114 N 14 O 31 (m / z) [M + H] calcd for ⁺ 1623.7847, found 1623.7780.

(10), (11), and (12), Alexa fluor (R) 647 conjugate
Alexa Fluor® 647 carboxylic acid succinimidyl ester was from Invitrogen (catalog number A-20106). The molecular weight was reported by Invitrogen to be about 1250. The molecular weight of the Alexa647-labeled compound was estimated based on [M + H] ⁺ measured value of 955.07 of Alexa Fluor 647 carboxylic acid succinimidyl ester from LCMS. The extinction coefficient for λ _max 650 is approximately 270000 ± 20000, which varies from batch to batch.

General procedure for HPLC purification:
Preparative high performance liquid chromatography (HPLC) uses a Waters XBridge BEH C18 OBD preparative column, 130Å, 5 μm, 19 mm × 100 mm (Waters, part no. 18620278), eluting with a linear gradient at a flow rate of 17 mL / min. I went. Solvent gradient: acetonitrile / water / trifluoroacetic acid (2: 98: 0.1) to (22: 78: 0.1) over 40 minutes. The collected fractions were analyzed by analytical LCMS and those determined to have sufficient purity were pooled and evaporated.

(10):
To a solution of compound (7) (3.0 mg, 4.8 μmol) in dimethyl sulfoxide (200 μL), Alexa Fluor® 647 carboxylic acid succinimidyl ester (5.0 mg, 4 μmol) and N , N-diisopropylethylamine (10 microL, 10 eq) was added. The reaction mixture was shaken for 1 hour at room temperature and then purified directly by preparative HPLC. The collected fractions were analyzed by analytical LCMS and those determined to have sufficient purity were pooled (R _t = 22.7-24 min). 3.2 mg of (10) was obtained (55% yield). The solution was aliquoted, evaporated in a vacuum centrifuge and the product stored at 4 ° C. MS (ESI) Calculated (m / z) for [M + H] ⁺ (m / z) about 1456, found 1456.82.

(11):
To a solution of compound (8) (6.0 mg, 5 μmol) in dimethyl sulfoxide (200 μL), Alexa Fluor® 647 carboxylic acid succinimidyl ester (5.0 mg, 4 μmol) and N, N -Diisopropylethylamine (10 microL, 10 eq) was added. The reaction mixture was shaken for 1 hour at room temperature and then purified directly by preparative HPLC. The collected fractions were analyzed by analytical LCMS and those determined to have sufficient purity were pooled (R _t = 25.3-36.7 min). 4.8 mg of (11) was obtained (62% yield). The solution was aliquoted, evaporated in a vacuum centrifuge and the product stored at 4 ° C. MS (ESI) Calculated for [M + H] ⁺ (m / z) about 1958, found 1958.74.

(12):
To a solution of compound (9) (9.8 mg, 6 μmol) in dimethyl sulfoxide (200 μL), Alexa Fluor® 647 carboxylic acid succinimidyl ester (5.0 mg, 4.8 μmol) and N , N-diisopropylethylamine (10 microL, 10 eq) was added. The reaction mixture was shaken for 1 hour at room temperature and then purified directly by preparative HPLC. The collected fractions were analyzed by analytical LCMS and those determined to have sufficient purity were pooled (R _t = 27.7 min). 5.2 mg of (12) was obtained (52% yield). The solution was aliquoted, evaporated in a vacuum centrifuge and the product stored at 4 ° C. Calculated value (m / z) for MS (ESI) [M + H] ^{+ is} about 2460, found value 2461.18.

4-amino-N- [1,31-bis (1-{[(1S, 2R, 3R, 4R, 5S) -4- (acetylamino) -2,3-dihydroxy-6,8-dioxabicyclo [ 3.2.1] oct-1-yl] methyl} -1H-1,2,3-triazol-4-yl) -2,6,10,14,18,22,26,30-octaoxahentria Contan-16-yl] butanamide (13) and 4-amino-N- {1,31-bis (1-{[(1S, 2R, 3R, 4R, 5S) -4- (acetylamino) -2,3 -Dihydroxy-6,8-dioxabicyclo [3.2.1] oct-1-yl] methyl} -1H-1,2,3-triazol-4-yl) -16- [15- (1- { [(1S, 2R, 3R, 4R, 5S) -4- (acetylamino) -2,3- Hydroxy-6,8-dioxabicyclo [3.2.1] oct-1-yl] methyl} -1H-1,2,3-triazol-4-yl) -2,6,10,14-tetraoxa Pentadec-1-yl] -2,6,10,14,18,22,26,30-octaoxahentriacontan-16-yl} butanamide (14)

Compound (Ie-1) (247 mg, 0.904 mmol) was dissolved in dichloromethane (15 mL) and pyridine was added (1.46 mL, 18.1 mmol). The reaction mixture was cooled at −20 ° C., a solution of trifluoromethanesulfonic anhydride (0.23 mL, 1.4 mmol) in dichloromethane (0.6 mL) was added dropwise and the mixture was warmed to 0 ° C. over 50 minutes. While stirring. The reaction mixture was diluted with dichloromethane and washed with 1M aqueous hydrogen chloride, saturated aqueous sodium bicarbonate, and saturated aqueous sodium chloride. The organic phase was dried over magnesium sulfate, filtered and concentrated. This crude material was used in the next step without further purification. Sodium azide (270 mg, 4.1 mmol) was added to the above triflate in dimethylformamide (4.1 mL). The reaction mixture was stirred at 60 ° C. for 16 hours. The solvent was evaporated and the crude material was purified through silica gel by flash chromatography (15/1 ethyl acetate / methanol) to give the desired compound (Ie-3) as a yellow oil (227 mg, 92% yield). ). [Α] _D 127 (c 1, methanol); ¹ H NMR (500 MHz, chloroform-d) delta ppm 1.34 (s, 3H), 1.53 (s, 3H), 2.00 (s, 3H ), 3.67 (d, J = 12.7 Hz, 1H), 3.72 (d, J = 7.8 Hz, 1H), 3.74 (d, J = 7.8 Hz, 1H), 3.75 (d, J = 12.7 Hz, 1H), 4.02-4.10 (m, 2H), 4.11 (d, J = 5.9 Hz, 1H), 5.35 (d , J = 2.4 Hz, 1H), 5.95 (d, J = 8.8 Hz, 1H); ¹³ C NMR (125 MHz, chloroform-d) delta ppm 23.2, 26.2, 27. 7, 51.0, 54.2, 69.3, 74.8, 76.1, 80.6, 101. , 111.6, 170.1; HRMS (ESI ) C 12 H 18 N 4 O 5 (m / z) [M + H] calcd for ⁺ 299.1350, found 299.1344.

Compounds (I-j-2) and (I-j-3) are propargyl bromide, (I-i-2) (commercially available from Dalton Pharma; DC-001760) and (I-i-3), respectively. ) (B. Ernst et al. In Bioorganic & Medicinal Chemistry, 16, 5216 (2008)) could be made following the same procedure described for the formation of compound (If-2).

Compound (Ij-2): Purification conditions: 20% ethyl acetate / hexane, 85 mg, oil (yield 8%); ¹ H NMR (400 MHz, chloroform-d / TMS) delta ppm 1.77-1. 90 (m, 14 H), 2.23 (t, J = 7.1 Hz, 2H), 2.43 (t, J = 2.3 Hz, 2H), 3.20-3.29 (m, 2H), 3.39-3.55 (m, 24H), 3.60 (t, J = 6.3 Hz, 4H), 4.13 (d, J = 2.5 Hz, 4H), 4. 15-4.24 (m, 1H), 5.09 (s, 2H), 5.16 (br.s., 1H), 6.05 (d, J = 8.1 Hz, 1H), 7. 28 - 7.41 (m, 5H) ; 13 C NMR (100 MHz, chloroform -d / TMS) delta pp 25.8, 29.8 (2C), 29.9 (2C), 30.0 (2C), 33.7, 40.4, 48.5, 58.1 (2C), 66.6, 67. 2 (2C), 67.6 (2C), 67.7 (2C), 67.8 (2C), 67.9 (2C), 68.3 (2C), 69.0 (2C), 74.2 (2C), 79.9 (2C) , 128.1, 128.5 (4C), 136.6, 156.6, 172.1; HRMS (ESI) C 39 H 62 N 2 O 11 (m / z ) Calculated value 735.4426, measured value 735.4424 for [M + H] ⁺ .

Compound (I-j-3): Purification conditions: 85% ethyl acetate / hexane, 32.6 mg, oil, (yield 71%); ¹ H NMR (400 MHz, chloroform-d / TMS) delta ppm 1.75 -1.90 (m, 20H), 2.18 (t, J = 6.9 Hz,
2H), 2.43 (t, J = 2.4 Hz, 3H), 3.23 (q, J = 6.3)
Hz, 2H), 3.40-3.53 (m, 30H), 3.59 (t, J = 6.3 Hz, 6H), 3.67 (s, 6H), 4.13 (d, J = 2.3 Hz, 6H), 5.08 (s, 2H), 5.27 (br.s., 1H), 5.85 (s, 1H), 7.27-7.40 (m, 5H) ); ¹³ C NMR (100 MHz, chloroform-d / TMS) delta ppm 25.7, 29.7 (3C), 29.9 (3C), 30.0 (3C), 34.4, 40.4, 58.1 (3C), 59.8, 66.5, 67.1 (3C), 67.6 (3C), 67.7 (3C), 67.8 (3C), 67.82 (3C), 68.4 (3C), 69.1 (3C), 74.2 (3C), 79.9 (3C), 128.0, 1 8.4 (4C), 136.6, 156.6 , 172.3; HRMS (ESI) C 52 H 84 N 2 O 15 (m / z) [M + H] calcd for ⁺ 977.5944, found Value 977.5943.

Compound (Ik-2):
THPTA (22.6 mg, 0.052 mmol) and copper sulfate (2.5 mg, 0.01 mmol) were dissolved in water (200 microL) and then (Ie-3) (45 mg, 0.152 mmol) and ( I-j-2) (51 mg, 0.069 mmol) was added to a methanol (1.1 mL) solution. Next, sodium ascorbate (4.2 mg, 0.021 mmol) dissolved in water (100 microL) was added and the reaction mixture was stirred at 50 ° C. for 24 hours. The solvent was evaporated and the crude material was purified through silica gel by flash chromatography (5% methanol in dichloromethane) to give the desired compound (Ik-2) as an oil (72 mg, 78% yield). ¹ H NMR (400 MHz, methanol-d ₄ ) delta ppm 1.34 (s, 6H), 1.52 (s, 6H), 1.73-1.86 (m, 12H), 1.97 (s 6H), 2.24 (t, J = 7.4 Hz, 2H), 3.15 (t, J = 6.8 Hz, 2H), 3.43-3.54 (m, 22H), 3 .58 (t, J = 6.3 Hz, 4H), 3.86 (d, J = 8.1 Hz, 2H), 3.97 (dd, J = 6.2, 1.9 Hz, 2H) , 4.11-4.23 (m, 5H), 4.58 (s, 4H), 4.78 (s, 6H), 4.91 (d, J = 14.1 Hz, 2H), 4. 98 (d, J = 14.4 Hz, 2H), 5.07 (s, 2H), 5.24 (d, J = 1.8 Hz, 2H), 7.2 5-7.40 (m, 5H), 7.9 (s, 2H); ¹³ C NMR (100 MHz, methanol-d ₄ ) delta ppm 22.7 (2C), 26.8 (2C), 27. 5, 28.4 (2C), 31.1 (4C), 31.2 (2C), 34.5, 41.4, 50.6, 51.0 (2C), 56.3 (2C), 64 .8 (2C), 67.5 (2C), 68.7 (2C), 68.8 (2C), 68.9 (3C), 69.0 (2C), 69.4 (2C), 69. 7 (2C), 70.9 (2C), 76.3 (2C), 76.6 (2C), 81.5 (2C), 102.5 (2C), 112.8 (2C), 127.2 (2C), 129.0 (2C), 129.1, 129.6 (2C), 138.6, 146.3 (2C), 159.0, 173.5 ( 2C), 175.5; HRMS (ESI) C 63 H 98 N 10 O 21 (m / z) [M + H] calcd for ⁺ 1331.6981, Found Value 1331.6971.

Compound (Ik-3):
THPTA (16 mg, 0.037 mmol) and copper sulfate (1.7 mg, 0.007 mmol) were dissolved in water (100 microL), then (Ie-3) (32.5 mg, 0.109 mmol) and ( I-j-3) (32 mg, 0.033 mmol) was added to a methanol (1.1 mL) solution. Next, sodium ascorbate (3 mg, 0.015 mmol) dissolved in water (100 microL) was added and the reaction mixture was stirred at 50 ° C. for 24 hours. The solvent was evaporated and the crude material was purified through silica gel by flash chromatography (10% methanol in dichloromethane) to give the desired compound (Ik-3) as an oil (43.5 mg, 70% yield). ). ¹ H NMR (400 MHz, methanol-d ₄ ) delta ppm 1.33 (s, 9H), 1.52 (s, 9H), 1.71-1.87 (m, 18H), 1.97 (s 9H), 2.20 (t, J = 7.3 Hz, 2H), 3.15 (t, J = 6.8 Hz, 2H), 3.43-3.52 (m, 28H), 3 .58 (t, J = 6.3 Hz, 6H), 3.67 (s, 6H), 3.86 (d, J = 8.3 Hz, 3H), 3.97 (dd, J = 6. 0, 1.8 Hz, 3H), 4.14-4.22 (m, 5H), 4.58 (s, 6H), 4.78 (s, 8H), 4.91 (d, J = 14 .6 Hz, 3H), 4.97 (d, J = 14.6 Hz, 3H), 5.07 (s, 2H), 5.24 (d, J = 2) 0 Hz, 3H), 7.26 - 7.38 (m, 5H), 7.98 (s, 3H); 13 C NMR (100 MHz, methanol -d ₄₎ delta ppm 22.7 (3C), 26 .9 (3C), 27.7, 28.4 (3C), 31.1 (3C), 31.2 (3C), 31.3 (3C), 35.2, 41.3, 51.0 ( 3C), 56.3 (3C), 61.8, 64.9 (3C), 67.5 (3C), 68.7 (3C), 68.8 (3C), 68.9 (3C), 69 0.0 (4C), 69.6 (3C), 69.7 (3C), 70.0 (3C), 76.3 (3C), 76.6 (3C), 81.5 (3C), 102. 5 (3C), 112.8 (3C), 127.2 (3C), 129.0 (2C), 129.1, 29.7 (2C), 138.6, 146.4 (3C), 159.0, 173.5 (3C), 175.6; HRMS (ESI) C 88 H 138 N 14 O 30 (m / z) Calculated value 1871.9776, measured value 1871.9713 for [M + H] ⁺

Compound (Ik-2) or (Ik-3) (0.068 mmol) was added to a mixture of acetic acid, methanol, and water (2.5-3 mL, 0.6-0.9 mL, 0.6 -0.9 mL) and stirred at 70 ° C. for 24 hours. The solvent was evaporated and the residue was coevaporated twice with toluene. The resulting crude material was used in the next step without further purification.

In a round bottom flask, compound (I-2) or (I-1-3) (1 equivalent) was dissolved in methanol (0.01 M) and the flask was flushed with nitrogen. Palladium on carbon (10%, 0.7 eq) was added and the flask was flushed with nitrogen and then with hydrogen. The reaction mixture was stirred at room temperature for 24 hours under a hydrogen atmosphere (using a balloon filled with hydrogen). The palladium was filtered using 0.45 μm PTFE Acrodisc Cr and rinsed once with methanol. The solvent was evaporated.

(13):
This crude material was dissolved in 0.5 mL methanol / water (50:50) and injected onto an HPLC column. Preparative high performance liquid chromatography (HPLC) uses a Waters XBridge BEH C18 OBD preparative column, 130Å, 5 μm, 19 mm × 100 mm (Waters, part no. 18620278), eluting with a linear gradient at a flow rate of 17 mL / min. I went. Solvent gradient: acetonitrile / water / trifluoroacetic acid (2: 98: 0.1) to (22: 58: 0.1) over 40 minutes. The collected fractions were analyzed by analytical LCMS and the 34.7-35.6 min fractions determined to be of sufficient purity were pooled and evaporated to give 12.8 mg of (13) as an oil. (Yield 17% over 2 steps). ¹ H NMR (500 MHz, methanol-d ₄ ) delta ppm 1.73-1.87 (m, 12H), 1.88-1.96 (m, 2H), 1.98 (s, 6H), 2 .39 (t, J = 7.0 Hz, 2H), 2.98 (t, J = 7.4 Hz, 2H), 3.42 (d, J = 8.5 Hz, 2H), 3.45 −3.55 (m, 24H), 3.59 (t, J = 6.3 Hz, 4H), 3.71−3.75 (m, 4H), 3.77 (d, J = 8.3) Hz, 2H), 3.96-4.02 (m, 2H), 4.13-4.20 (m, 1H), 4.58 (s, 4H), 4.91-4.95 (m, 4H), 5.20 (d, J = 1.5 Hz, 2H), 7.98 (s, 2H); HRMS (ESI) C 49 H 84 N 10 _{19 (m / z) [M} + H] calcd for ⁺ 1117.5987, found 1117.5977.

(14):
The crude material was dissolved in 0.5 mL methanol / water (50:50) and injected onto an HPLC column. Preparative high performance liquid chromatography (HPLC) uses a Waters XBridge BEH C18 OBD preparative column, 130Å, 5 μm, 19 mm × 100 mm (Waters, part no. 18620278), eluting with a linear gradient at a flow rate of 17 mL / min. I went. Solvent gradient: acetonitrile / water / trifluoroacetic acid (2: 98: 0.1) to (42: 58: 0.1) over 40 minutes. The collected fractions were analyzed by analytical LCMS and fractions 24.7-25.6 min determined to be of sufficient purity were pooled and evaporated to give 5.5 mg of (14) as an oil. (Yield 10% over 2 steps); ¹ H NMR (500 MHz, methanol-d ₄ ) delta ppm 1.75-1.86 (m, 18H), 1.87-1.94 (m, 2H) 1.98 (s, 9H), 2.37 (t, J = 6.8 Hz, 2H), 2.98 (t, J = 7.4 Hz, 2H), 3.43 (d, J = 8.5 Hz, 3H), 3.46-3.53 (m, 31H), 3.59 (t, J = 6.3 Hz, 6H), 3.68 (s, 6H), 3.72- 3.76 (m, 5H), 3.77 (d, J = 8.3 Hz, 3H), 3.97-4. 2 (m, 3H), 4.59 (s, 6H), 4.90-4.96 (m, 6H), 5.20 (d, J = 1.2 Hz, 3H) 7.98 (s, 3H); HRMS (ESI) C 71 H 120 N 14 O 28 (m / z) [M + H] calcd for ⁺ 1617.8469, found 1617.8415.

(15) and (16), Alexa fluor® 647 (AF647) conjugate
Alexa Fluor® 647 carboxylic acid succinimidyl ester was from Invitrogen (catalog number A-20106). The molecular weight was reported by Invitrogen to be about 1250. The molecular weight of the Alexa647-labeled compound was estimated based on the [M + H] ⁺ measured value of 955.07 of Alexa Fluor 647 carboxylic acid succinimidyl ester from LCMS. The extinction coefficient for λ _max 650 is approximately 270000 ± 20000, which varies from batch to batch.

General procedure for HPLC purification:
Preparative high performance liquid chromatography (HPLC) uses a Waters XBridge BEH C18 OBD preparative column, 130Å, 5 μm, 19 mm × 100 mm (Waters, part no. 18620278), eluting with a linear gradient at a flow rate of 17 mL / min. I went. Solvent gradient: acetonitrile / water / trifluoroacetic acid (2: 98: 0.1) to (22: 78: 0.1) over 40 minutes. The collected fractions were analyzed by analytical LCMS and those determined to have sufficient purity were pooled and evaporated.

(15):
To a solution of compound (13) (4.5 mg, 4 μmol) in dimethyl sulfoxide (200 μL), Alexa Fluor® 647 carboxylic acid succinimidyl ester (5.0 mg, 4 μmol) and N, N -Diisopropylethylamine (10 microL, 10 eq) was added. The reaction mixture was shaken for 1 hour at room temperature and then purified directly by preparative HPLC. The collected fractions were analyzed by analytical LCMS and those determined to have sufficient purity were pooled (R _t = 37.3-39 min). 4.0 mg of (15) was obtained (yield 50%). The solution was aliquoted, evaporated in a vacuum centrifuge and the product stored at 4 ° C. MS (ESI) Calculated for [M + H] ⁺ (m / z) about 1955, found 1955.32.

(16):
To a solution of compound (14) (5.2 mg, 3.2 μmol) in dimethyl sulfoxide (200 μL), Alexa Fluor® 647 carboxylic acid succinimidyl ester (5.0 mg, 4 μmol) and N , N-diisopropylethylamine (10 microL, 10 eq) was added. The reaction mixture was shaken for 1 hour at room temperature and then purified directly by preparative HPLC. The collected fractions were analyzed by analytical LCMS, and those determined to have sufficient purity were pooled (R _t = 24.3-25.3 min). 4.0 mg of (16) was obtained (51% yield). The solution was aliquoted, evaporated in a vacuum centrifuge and the product stored at 4 ° C. MS (ESI) Calculated (m / z) for [M + H] ⁺ (m / z) approx. 2455, found 2456.90.

Basic alkylation / deprotection conditions to obtain (17)-(21):
The desired iodoalkyl, tetrabutylammonium hydrogen sulfate, and 12.5 M aqueous sodium hydroxide solution were added to a dichloromethane solution of (Ie-1). The reaction mixture was stirred at room temperature overnight, diluted with water and dichloromethane, and the aqueous phase was extracted twice more with dichloromethane. The combined organic layers were washed with 1M aqueous hydrochloric acid, water, dried over magnesium sulfate, filtered and concentrated under reduced pressure. The resulting material may be crude and may be subsequently subjected to the next reaction or may be purified through silica gel using flash chromatography. The resulting material was dissolved in a mixture of acetic acid / methanol / water (3: 1: 1 v / v) and the solution was heated to 60-70 ° C. overnight. The reaction mixture was concentrated under reduced pressure, coevaporated twice with toluene, and the crude material was purified by flash chromatography through silica gel or by reverse phase chromatography.

N-[(1S, 2R, 3R, 4R, 5S) -1- (ethoxymethyl) -2,3-dihydroxy-6,8-dioxabicyclo [3.2.1] oct-4-yl] acetamide ( 17)
(17) was synthesized as described in the general procedure above using iodoethane (20 eq). This crude product was dissolved in methanol, and activated carbon was added thereto. The mixture was stirred for 15 minutes, filtered and concentrated under reduced pressure. The crude material was purified by flash chromatography through silica gel eluting with ethyl acetate / methanol (15: 1). Fractions containing the desired product were collected and concentrated under reduced pressure. To this crude material was added ethyl acetate / methanol (15: 1) to give a precipitate which was filtered to give 9.1 mg (32% yield) of the desired product as a solid. ¹ H NMR (400 MHz, methanol-d ₄ ) delta ppm 5.23 (d, J = 1.5 Hz, 1 H), 3.97 (dd, J = 9.7, 1.4 Hz, 1 H ), 3.94 (d, J = 9.3 Hz, 1 H), 3.88 (d, J = 4.3 Hz, 1 H), 3.79 (d, J = 8.1 Hz, 1 H), 3.73 (dd, J = 9.8, 4.3 Hz, 1 H), 3.66 (d, J = 8.1 Hz, 1 H), 3.60 (d, J = 9 .6 Hz, 1 H), 3.59 (dq, J = 9.6, 7.1 Hz, 1 H), 3.55 (dq, J = 9.6, 7.1 Hz, 1 H), 2.00 (s, 3 H), 1.19 (t, J = 6.9 Hz, 3 H). LCMS (APCI) m / z: 262.1 [M + H] (100%).

N-[(1S, 2R, 3R, 4R, 5S) -2,3-dihydroxy-1- (propoxymethyl) -6,8-dioxabicyclo [3.2.1] oct-4-yl] acetamide ( 18)
(18) was synthesized as described in the general procedure above using iodopropane (20 eq). The crude product was purified using flash chromatography through silica gel eluting with ethyl acetate / methanol (15: 2) to give 13.9 mg (80% yield) of the desired product as an oil. . ¹ H NMR (400 MHz, methanol-d ₄ ) delta ppm 5.23 (d, J = 1.5 Hz, 1 H), 3.97 (dd, J = 9
. 7, 1.4 Hz, 1 H), 3.95 (d, J = 9.6 Hz, 1 H), 3.89 (d, J = 4.3 Hz, 1 H), 3.79 (d , J = 7.8 Hz, 1 H), 3.73 (dd, J = 9.8, 4.3 Hz, 1 H), 3.66 (d, J = 8.1 Hz, 1 H), 3.59 (d, J = 9.3 Hz, 1 H), 3.49 (dt, J = 9.3, 6.5 Hz, 1 H), 3.46 (dt, J = 9.3) 6.5 Hz, 1 H), 2.01 (s, 3 H), 1.60 (qt, J = 7.4, 6.5 Hz, 2 H), 0.94 (t, J = 7. 4 Hz, 3 H). LCMS (APCI) m / z: 276.2 [M + H] (100%).

N-[(1S, 2R, 3R, 4R, 5S) -1- (butoxymethyl) -2,3-dihydroxy-6,8-dioxabicyclo [3.2.1] oct-4-yl] acetamide ( 19)
(19) was synthesized using iodobutane (20 eq) as described in the general procedure above. The desired crude product was purified using flash chromatography through silica gel eluting with ethyl acetate / methanol (15: 1) to give 18 mg (100% yield) of the desired product as an oil. ¹ H NMR (400 MHz, methanol-d ₄ ) delta ppm 5.23 (d, J = 1.3 Hz, 1 H), 3.97 (dd, J = 9.6, 1.3 Hz, 1 H ), 3.94 (d, J = 9.6 Hz, 1 H), 3.88 (d, J = 4.3 Hz, 1 H), 3.79 (d, J = 7.8 Hz, 1 H), 3.73 (dd, J = 9.8, 4.3 Hz, 1 H), 3.66 (d, J = 7.8 Hz, 1 H), 3.59 (d, J = 9 .3 Hz, 1 H), 3.54 (dt, J = 9.3, 6.5 Hz, 1 H), 3.50 (dt, J = 9.3, 6.5 Hz, 1 H), 2.00 (s, 3 H), 1.52-1.61 (m, 2 H), 1.34-1.45 (m, 2 H), 0.95 (t, J = 7.3 Hz , 3 H). LCMS (APCI) m / z: 290.2 [M + H] (100%).

N-{(1S, 2R, 3R, 4R, 5S) -2,3-dihydroxy-1-[(pentyloxy) methyl] -6,8-dioxabicyclo [3.2.1] oct-4-yl } Acetamide (20)
(20) was synthesized as described in the general procedure above using iodopentane (20 eq). The desired crude product was purified using flash chromatography through silica gel eluting with ethyl acetate / methanol (15: 1) to give 17 mg (68% yield) of the desired product as an oil. ¹ H NMR (400 MHz, methanol-d ₄ ) delta ppm 5.23 (d, J = 1.5 Hz, 1 H), 3.97 (dd, J = 9.8, 1.3 Hz, 1 H ), 3.94 (d, J = 9.6 Hz, 1 H), 3.88 (d, J = 4.3 Hz, 1 H), 3.79 (d, J = 8.1 Hz, 1 H), 3.73 (dd, J = 9.8, 4.3 Hz, 1 H), 3.66 (d, J = 8.1 Hz, 1 H), 3.59 (d, J = 9 .6 Hz, 1 H), 3.53 (dt, J = 9.3, 6.5 Hz, 1 H), 3.49 (dt, J = 9.3, 6.5 Hz, 1 H), 2.01 (s, 3 H), 1.53-1.63 (m, 2 H), 1.29-1.41 (m, 4 H), 0.89-0.97 (m, 3 H ). LCMS (APCI) m / z: 304.1 [M + H] (100%).

N-{(1S, 2R, 3R, 4R, 5S) -1-[(hexyloxy) methyl] -2,3-dihydroxy-6,8-dioxabicyclo [3.2.1] oct-4-yl } Acetamide (21)
(21) was synthesized as described in the general procedure above using iodohexane (20 eq). The desired crude product was purified using flash chromatography through silica gel eluting with ethyl acetate / methanol (15: 1) to give 56 mg of product as an oil. This material was re-purified using reverse phase chromatography to give 7.1 mg (15% yield) of the desired product as a solid. ¹ H NMR (400 MHz, methanol-d ₄ ) delta ppm 5.23 (d, J = 1.5 Hz, 1 H), 3.96 (dd, J = 10.1, 1.3 Hz, 1 H ), 3.94 (d, J = 9.6 Hz, 1 H), 3.88 (d, J = 4.3 Hz, 1 H), 3.79 (d, J = 7.8 Hz, 1 H), 3.73 (dd, J = 9.8, 4.3 Hz, 1 H), 3.66 (d, J = 8.1 Hz, 1 H), 3.59 (d, J = 9 .6 Hz, 1 H), 3.53 (dt, J = 9.3, 6.5 Hz, 1 H), 3.49 (dt, J = 9.3, 6.5 Hz, 1 H), 2.00 (s, 3 H), 1.53-1.62 (m, 2 H), 1.27-1.50 (m, 6 H), 0.89-0.97 (m, 3 H ). LCMS (APCI) m / z: 318.1 [M + H] (100%).

N-[(1S, 2R, 3R, 4R, 5S) -2,3-dihydroxy-1- (2,5,8,11,14-pentaoxapentadec-1-yl) -6,8-dioxa Bicyclo [3.2.1] oct-4-yl] acetamide (22)
To a solution of (Ie-1) in dichloromethane (3 mL), pyridine (0.3 mL, 4 mmol) is added, the mixture is cooled to −20 ° C., and trifluoromethanesulfonic anhydride (0.047 mL, .0. 28 mmol) in dichloromethane (0.6 mL) was added. The reaction mixture was warmed to −10 ° C. over 1 hour, diluted with dichloromethane, washed successively with 1M aqueous hydrochloric acid, saturated aqueous sodium bicarbonate, brine, dried over magnesium sulfate, filtered, and reduced pressure Concentrated under to give the desired crude material that was used in the next step without further purification. To a solution of 2,5,8,11-tetraoxatridecan-13-ol (207 mg, 0.994 mmol) cooled to 0 ° C. in N, N-dimethylformamide was added sodium hydride (39.9 mg, 1.0 mmol). Was added and the reaction mixture was stirred for 10 minutes. The above crude ((3aR, 4R, 7S, 8R, 8aR) -8-acetamido-2,2-dimethyltetrahydro-4,7-epoxy [1,3] dioxolo [4,5-d] oxepin-4 (5H ) -Yl) methyl trifluoromethanesulfonate in N, N-dimethylformamide (0.5 mL) was added dropwise and the reaction mixture was stirred at 0 ° C. for 25 min. The reaction was quenched with methanol and the reaction mixture was stirred for 5 minutes. The resulting mixture was then concentrated under reduced pressure and the resulting residue was dissolved in dichloromethane and washed with water. The aqueous layer was extracted twice more with dichloromethane. The combined organic layers were washed with water and concentrated under reduced pressure. The crude material was purified by flash chromatography through silica gel eluting with ethyl acetate / methanol (15: 2) to give 85 mg (100%) of the desired product. N-((3aR, 4S, 7S, 8R, 8aR) -2,2-dimethyl-4- (2,5,8,11,14-pentaoxapentadecyl) hexahydro-4,7-epoxy [1,3 ] A solution of dioxolo [4,5-d] oxepin-8-yl) acetamide (85 mg, 0.18 mmol) in acetic acid / methanol / water (3.9: 1.3: 1.3 v / v) Heated to ° C overnight. The reaction mixture is concentrated under reduced pressure and the resulting crude material is evaporated twice with toluene and purified by flash chromatography through silica gel, eluting with 10% methanol / dichloromethane to yield 15 mg of the desired product ( 22) was obtained as an oil. ¹ H NMR (400 MHz, methanol-d ₄ ) delta ppm 5.22 (d, J = 1.3 Hz, 1 H), 3.96 (d, J = 9.6 Hz, 1 H), 95 (dd, J = 9.9, 1.3 Hz, 1 H), 3.89 (d, J = 4.3 Hz, 1 H), 3.78 (d, J = 8.1 Hz, 1 H), 3.59-3.74 (m, 17 H), 3.53-3.56 (m, 2 H), 3.36 (s, 3 H), 1.99 (s, 3 H) . LCMS (APCI) m / z: 424.2 [M + H] (13%), 441.3 [M + NH 4] (100%).

(1R, 2R, 3R, 4R, 5S) -4-acetamido-1-(((4-bromobenzoyl) oxy) methyl) -6,8-dioxabicyclo [3.2.1] octane-2,3 -Diylbis (4-bromobenzoate) (23)
To a solution of (3) (9 mg) in anhydrous N, N-dimethylformamide (500 μL) cooled at room temperature, N, N-diisopropylethylamine (34 μL) and 4- (dimethylamino) pyridine (4.3 mg) Followed by p-bromobenzoyl chloride (44 mg) and the resulting mixture was stirred at room temperature for 4.5 hours. Water was added, the resulting mixture was extracted three times with ethyl acetate, and the combined organic phases were washed successively with 0.5M aqueous hydrochloric acid and brine. The organic phase is dried over magnesium sulfate, filtered and concentrated and the crude material is purified by flash chromatography through silica gel eluting with a gradient of 0-100% ethyl acetate in heptane to yield 23 mg of product. (23) was obtained (yield 80%).
1H NMR (400 MHz, CDCl3): delta (ppm) 7.89-7.95 (m, 2H), 7.78-7.84 (m, 2H), 7.54-7.66 (m , 6 H), 7.41-7.46 (m, 2 H), 5.87 (d, J = 8.8 Hz, 1 H), 5.80 (d, J = 4.3 Hz, 1 H), 5.60 (d, J = 1.l Hz, 1 H), 5.44 (dd, J = 10.2, 4.5 Hz, 1 H), 4.54-4.64 (m , 3 H), 4.15 (d, J = 8.6 Hz, 1 H), 3.93 (d, J = 8.6 Hz, 1 H), 1.95 (s, 3 H). ¹³ C NMR (101 MHz, CDCl 3) Delta ppm 170.6, 165.6, 165.0, 164.9, 132.1, 131.9, 131.8, 131.4, 131.2, 131.2 , 129.3, 129.0, 128.9, 127.7, 127.5, 127.4, 101.8, 81.6, 69.5, 68.8, 68.4, 62.5, 52 .7, 23.2. Single crystals were obtained by the vapor diffusion technique using methanol and heptane as solvents. Single crystal X-ray analysis: Data collection was performed on a Bruker APEX diffractometer at room temperature. Data collection consisted of 3 omega scans at low angles and 3 high angles each with 0.5 steps. In addition, two phi scans were collected to improve the quality of the absorption correction. The structure is non-faceless twins and refined by ignoring the second domain. The structure was elucidated by a direct method using the SHELX software suite (see SHELXTL, Version 5.1, Bruker AXS, 1997) in space group P2 (1). After that, the structure was refined by the perfect matrix least squares method. Anisotropic displacement parameters were used to find and refine all non-hydrogen atoms. The hydrogen atom on the nitrogen was placed at this position and constrained to a reasonable position. The remaining hydrogen atoms were placed at the calculated positions and placed on their carrier atoms. The final refinement included isotropic displacement parameters for all hydrogen atoms. Analysis of absolute structure using the likelihood method (RWW Hooft et al. J. Appl. Cryst., 41, 96-103 (2008)) was performed using PLATON (AL Spek, J. Appl. Cryst., 36, 7-13 (2003)). The result shows that the absolute structure was assigned correctly. This method calculates that the probability that the structure is correct is 100.0. The Foot parameter is reported as esd 0.013 at 0.036. In addition, the Flack parameter is 0.03 and esd 0.04. The final R index was 5.6%. The final difference Fourier revealed no missing or misplaced electron density. The relevant crystals, data collection and refinement are summarized in Table 1 and FIG.
Table 1. Crystal data and structural refinement for (23).
Experimental formula C15H12Br1.50N0.50O4.50
Formula weight 391.12
Temperature 296 (2) K
Wavelength 1.54178Å
Crystalline monoclinic space group P2 (1)
Unit cell dimensions a = 12.5748 (9) Å α = 90 °
b = 5.6465 (4) Åβ = 97.453 (4) °
c = 21.2806 (16) Å γ = 90 °
Volume 1498.23 (19) Å 3
Z 4
Density (calculated value) 1734 Mg / m ³
Absorption coefficient 5.476mm ^-1
F (000) 776
Crystal size 0.37 × 0.22 × 0.15mm ³
Theta range for data collection 2.09-68.30 °
Exponential range −13 ≦ h ≦ 5, −5 ≦ k ≦ 6, −24 ≦ 1 ≦ 24
Collected reflections 8050
Independent reflection 4408 [R (int) = 0.0247]
Theta = 68.30 ° completeness 93.9%
Absorption correction Empirical refinement method F2 complete matrix least squares data / suppression / parameter 4408/32/389
F2 fitness 1.031
Final R index [I> 2 Sigma (I)] R1 = 0.0563, wR2 = 0.1658
R index (all data) R1 = 0.0589, wR2 = 0.701
Absolute structural parameter 0.04 (3)
Maximum difference peak and hole 0.949 and -0.685 e. Å ^-3

(1S, 2R, 3R, 4R, 5S) -4-Azido-2,3-bis (benzyloxy) -1-[(benzyloxy) methyl] -6,8-dioxabicyclo [3.2.1] Octane (l-m-1)
(1S, 2R, 3R, 4R, 5S) -4-azido-1- (hydroxymethyl) -6,8-dioxabicyclo [3.2.1] octane-2,3-diol (1) (445 mg, To a solution of 2.05 mmol) N, N-dimethylformamide (5 mL) was added sodium hydride (60% mineral oil dispersion, 410 mg, 10.2 mmol) at room temperature. The reaction became very thick and could not be sufficiently stirred. An additional 5 mL of N, N-dimethylformamide was added and the reaction was stirred at room temperature for 30 minutes before benzyl bromide (1.23 mL, 10.2 mmol) was added dropwise. The reaction was stirred overnight at room temperature. The next morning, the reaction was quenched with water and extracted three times with ethyl acetate. The combined organic layers were washed with water, brine, dried over sodium sulfate, filtered and concentrated under reduced pressure. The crude material was purified using CombiFlash Rf (RediSep 40 g silica gel column) eluting with an ethyl acetate / heptane gradient from 0% to 30% to give the title compound (890.0 mg, 89.1%). . Method C: Run for 3 minutes LRMS [M + Na = 510]. ¹ H NMR (methanol-d ₄ ) δ: 7.07-7.52 (m, 15H), 5.31 (s, 1H), 4.81 (d, J = 5.1 Hz, 1H), 4 .78 (d, J = 5.5 Hz, 1H), 4.68-4.74 (m, 1H), 4.47-4.51 (m, 1H), 4.46 (d, J = 6 .6 Hz, 1H), 4.35-4.42 (m, 1H), 4.12 (d, J = 3.9 Hz, 1H), 3.87-3.91 (m, 2H), 3 .86 (d, J = 8.2 Hz, 1H), 3.62 (d, J = 8.2 Hz, 1H), 3.58 (d, J = 9.4 Hz, 1H), 3.46 (D, J = 8.6 Hz, 1H)

(1S, 2R, 3R, 4R, 5S) -2,3-bis (benzyloxy) -1-[(benzyloxy) methyl] -6,8-dioxabicyclo [3.2.1] octane-4- Amine (1-n-1)
(1S, 2R, 3R, 4R, 5S) -4-Azido-2,3-bis (benzyloxy) -1-[(benzyloxy) methyl] -6,8-dioxabicyclo [3.2.1] A mixture of octane (1-m-1) (310 mg, 0.64 mmol), triphenylphosphine (334 mg, 1.27 mmol), water (92 mg, 5.1 mmol) and tetrahydrofuran (10 mL) was added at 65 ° C. for 16 hours. Stir. The reaction mixture was concentrated under reduced pressure and the residue was loaded onto a silica gel column. Chromatography eluting with a gradient of 20% to 80% ethyl acetate in heptane provided the title product as a colorless gum (210 mg, 72%). ¹ H NMR (chloroform-d) δ: 7.20-7.37 (m, 15H), 5.29 (d, J = 1.2 Hz, 1H), 4.90 (d, 11.5 Hz, 1H), 4.79 (d, J = 11.5 Hz, 1H), 4.57 (d, J = 11.7 Hz, 1H), 4.56 (d, J = 12.1 Hz, 1H), 4.43 (d, J = 12.1 Hz, 1H), 4.39 (d, J = 11.7 Hz, 1H), 3.97 (d, J = 3.9 Hz, 1H), 3.90. (D, J = 9.0 Hz, 1H), 3.69 (d, J = 8.2 Hz, 1H), 3.59 (d, J = 8.2 Hz, 1H), 3.42 (d , J = 8.6 Hz, 1H), 3.37 (dd, J = 9.4, 3.5 Hz, 1H), 3.07 (dd, J = 9.4, 1.2 Hz) , 1H); ¹³ C NMR (chloroform-d) δ: 131.8, 131.6, 131.5, 128.2, 128.2, 128.1, 128.1, 128.0, 127.7, 127.6, 127.6, 127.4, 103.5, 82.6, 80.3, 74.5, 73.3, 73.1, 72.2, 69.9, 69.3, 55. 1; LCMS (ES +): 1.18 min, 484.2 (M + Na) ⁺ .

N-{(1S, 2R, 3R, 4R, 5S) -2,3-bis (benzyloxy) -1-[(benzyloxy) methyl] -6,8-dioxabicyclo [3.2.1] octa -4-yl} acetamide (ln-2)
(1S, 2R, 3R, 4R, 5S) -2,3-bis (benzyloxy) -1-[(benzyloxy) methyl] -6,8-dioxabicyclo [3.2.1] octane-4- To a stirred mixture of amine (1-n-1) (25 mg, 0.054 mmol), pyridine (43 mg, 0.54 mmol) and 2-methyl-tetrahydrofuran (1 mL) was added acetic anhydride (46 mg, 0.43 mmol) at room temperature. At once. The reaction mixture was stirred at room temperature for 16 hours and partitioned between ethyl acetate and water. The organic extract was washed with brine, dried over anhydrous magnesium sulfate and concentrated under reduced pressure. The residue was purified on a silica gel column eluting with a gradient from 20% to 60% ethyl acetate in heptane to give the title product as a white solid (20 mg, 73%). ¹ H NMR (chloroform-d) δ: 7.24-7.43 (m, 15H), 5.35 (d, J = 1.2 Hz, 1H), 5.06 (d, J = 8.6) Hz, 1H), 4.96 (d, J = 11.3 Hz, 1H), 4.74 (d, J = 12.5 Hz, 1H), 4.58 (d, J = 11.3 Hz, 1H), 4.42 (d, J = 12.5 Hz, 1H), 4.40 (s, 2H), 4.30-4.36 (m, 1H), 4.04 (d, J = 3 .9 Hz, 1H), 3.96 (d, J = 8.6 Hz, 1H), 3.67-3.70 (m, 1H), 3.58-3.61 (m, 1H), 3 .41-3.47 (m, 2H), 1.87 (s, 3H); 13 C NMR ( chloroform -d) δ: 170.0, 138.2, 137.8 137.4, 128.7, 128.5, 128.4, 128.3, 128.2, 128.1, 128.0, 127.8, 101.6, 82.8, 75.7, 75. 0, 73.8, 73.2, 71.5, 70.1, 69.5, 53.5, 23.3; LCMS (ES +): 1.87 min, 526.3 (M + Na) ⁺ .

N-{(1S, 2R, 3R, 4R, 5S) -2,3-bis (benzyloxy) -1-[(benzyloxy) methyl] -6,8-dioxabicyclo [3.2.1] octa -4-yl} -2,2,2-trifluoroacetamide (1-n-3)
(1S, 2R, 3R, 4R, 5S) -2,3-bis (benzyloxy) -1-((benzyloxy) methyl) -6,8-dioxabicyclo [3.2.1] octane-4- To a stirred mixture of amine (1-n-1) (75 mg, 0.16 mmol), pyridine (129 mg, 1.62 mmol), and 2-methyl-tetrahydrofuran (1 mL) was added trifluoroacetic anhydride (102 mg, 0.49 mmol). Was added in one portion at room temperature. The reaction mixture was stirred at room temperature for 16 hours and partitioned between ethyl acetate and water. The organic extract was washed with brine, dried over anhydrous magnesium sulfate and concentrated under reduced pressure. The residue was purified on a silica gel column eluting with a gradient of 10% to 40% ethyl acetate in heptane to give the title product as a white solid (60 mg, 66%). ¹ H NMR (chloroform-d) δ:
7.25-7.42 (m, 15H), 5.91 (d, J = 8.6 Hz, 1H),
5.35 (d, J = 1.2 Hz, 1H), 4.95 (d, J = 11.3 Hz, 1H), 4.72 (d, J = 12.5 Hz, 1H), 4.58 (D, J = 11.3 Hz, 1H), 4.41 (s, 2H), 4.40 (d, J = 12.5 Hz, 1H), 4.36 (t, J = 9.8 Hz) , 1H), 4.08 (d, J = 3.9 Hz, 1H), 3.96 (d, J = 9.0 Hz, 1H), 3.68-3.72 (m, 1H), 3 .60-3.63 (m, 1H), 3.50 (dd, J = 10.0, 3.7 Hz, 1H), 3.45 (d, J = 8.6 Hz, 1H); ¹³ C NMR (chloroform-d) δ: 137.9, 137.1, 136.1, 128.9, 128.5, 128.5, 128.4, 128.4, 12 8.1, 128.1, 128.1, 127.9, 100.6, 83.0, 75.0, 74.9, 73.8, 72.7, 71.4, 70.3, 69. 2, 54.1; ¹⁹ F NMR (chloroform-d) δ: -75.7 (s); LCMS (ES-): 2.11 min, 556.2 (M−H) ⁻ .

N-{(1S, 2R, 3R, 4R, 5S) -2,3-bis (benzyloxy) -1-[(benzyloxy) methyl] -6,8-dioxabicyclo [3.2.1] octa -4-yl} methanesulfonamide (1-n-4)
(1S, 2R, 3R, 4R, 5S) -2,3-bis (benzyloxy) -1-((benzyloxy) methyl) -6,8-dioxabicyclo [3.2.1] octane-4- To a stirred mixture of amine (1-n-1) (50 mg, 0.11 mmol), triethylamine (0.100 mL, 0.72 mmol), and 2-methyl-tetrahydrofuran (1 mL) was added methanesulfonyl chloride (0.025 mL, 0 .33 mmol) was added dropwise at 0 ° C. The reaction mixture was stirred at room temperature for 16 hours and partitioned between ethyl acetate and saturated aqueous sodium bicarbonate. The organic extract was washed with water, brine, dried over anhydrous magnesium sulfate and concentrated under reduced pressure. The residue was purified on a silica gel column eluting with a gradient of 10% to 50% ethyl acetate in heptane to give the title product as a white solid (58 mg, 65%). ¹ H NMR (chloroform-d) δ: 7.22-7.41 (m, 15H), 5.43 (d, J = 1.2 Hz, 1H), 4.93 (d, J = 11.3) Hz, 1H), 4.79 (d, J = 11.7 Hz, 1H), 4.62 (br.s., 1H), 4.56 (d, J = 11.7 Hz, 1H), 4.54 (D, J = 11.3 Hz, 1H), 4.41 (d, J = 12.1 Hz, 1H), 4.37 (d, J = 12.1 Hz, 1H), 4.05 (d, J = 3.9 Hz, 1H), 3.92 (d, J = 8.6 Hz, 1H), 3.74 (d, J = 8.6 Hz, 1H), 3.68-3.72 ( m, 1H), 3.62 (d, J = 8.6 Hz, 1H), 3.54 (dd, J = 10.0, 3.7 Hz, 1H), 3.44 ( , J = 9.0 Hz, 1H) , 2.90 (s, 3H); 13 C NMR ( chloroform -d) δ: 138.0, 137.3, 137.2, 128.7 (2), 128 .5 (2), 128.4 (2), 128.3, 128.1 (2), 128.1, 127.9 (2), 127.8 (2), 102.7, 82.9, 77.2, 77.1, 75.0, 73.7, 73.5, 72.8, 70.2, 69.3, 57.7, 41.2 ;; LCMS (ES-): 1.97 Min, 538.3 (M−H) ⁻ .

N-{(1S, 2R, 3R, 4R, 5S) -2,3-bis (benzyloxy) -1-[(benzyloxy) methyl] -6,8-dioxabicyclo [3.2.1] octa -4-yl} propanamide (1-n-5)
To a stirred mixture of propionic acid (23 mg, 0.31 mmol) in tetrahydrofuran (1 mL), 1 mL of 1,1′-carbonyldiimidazole (33 mg, 0.20 mmol) was added in one portion at room temperature and the clear solution was added at room temperature 3 Stir for hours. Triethylamine (0.028 mL, 0.20 mmol) and (1S, 2R, 3R, 4R, 5S) -2,3-bis (benzyloxy) -1-((benzyloxy) methyl) -6,8-dioxabicyclo [3.2.1] Octane-4-amine (1-n-1) (47 mg, 0.10 mmol) was added in one portion at room temperature. The reaction mixture was stirred at room temperature for 16 hours and partitioned between ethyl acetate (3 mL), brine (2 mL) and water (2 mL). The organic extract was washed with brine, dried over anhydrous magnesium sulfate and concentrated under reduced pressure. The residue was purified on a silica gel column eluting with a gradient of 10% to 50% ethyl acetate in heptane to give the title product as a white solid (43 mg, 82%). ¹ H-NMR (chloroform-d) δ: 7.03-7.58 (m, 15H), 5.35 (s, 1H), 5.09 (d, J = 8.2 Hz, 1H), 4. 95 (d, J = 11.3 Hz, 1H), 4.72 (d, J = 12.1 Hz, 1H), 4.57 (d, J = 11.3 Hz, 1H), 4.43 (d, J = 12.1 Hz, 1 H), 4.39 (s, 2 H), 4.31-4.38 (m, 1 H), 4.04 (d, J = 3.9 Hz, 1 H), 3.95 (d , J = 9.0 Hz, 1H), 3.68 (d, J = 8.2 Hz, 1H), 3.59 (d, J = 8.2 Hz, 1H), 3.44-3.49 (m, 1H), 3.44 (d, J = 8.6 Hz, 1H), 2.08 (q, J = 7.4 Hz, 2H), 1.11 (t, J = 7.6 Hz, 3H) ). ¹³ C-NMR (chloroform-d) δ: 173.5, 138.1, 137.8, 137.4, 128.6, 128.5, 128.3, 128.3, 128.2, 128.1 , 128.0, 128.0, 127.7, 101.6, 82.7, 75.7, 74.9, 73.7, 73.2, 71.5, 70.1, 69.4, 53 .3, 29.6, 9.5. LCMS (ES-): 1.94 min, 516.4 (M-H) ^- . LCMS (AP +): 1.94 min, 518.5 (M + H) ⁺ .

N-{(1S, 2R, 3R, 4R, 5S) -2,3-bis (benzyloxy) -1-[(benzyloxy) methyl] -6,8-dioxabicyclo [3.2.1] octa -4-yl} -3,3,3-trifluoropropanamide (1-n-6)
To a stirred mixture of 3,3,3-trifluoropropionic acid (39 mg, 0.31 mmol) in tetrahydrofuran (1 mL) was added 1,1′carbonyldiimidazole (33 mg, 0.20 mmol) in one portion at room temperature. The clear solution was stirred at room temperature for 3 hours. Triethylamine (0.028 mL, 0.20 mmol) and (1S, 2R, 3R, 4R, 5S) -2,3-bis (benzyloxy) -1-((benzyloxy) methyl) -6,8-dioxabicyclo [3.2.1] Octane-4-amine (1-n-1) (47 mg, 0.10 mmol) was added in one portion at room temperature. The reaction mixture was stirred at room temperature for 16 hours and partitioned between ethyl acetate (3 mL), brine (2 mL) and water (2 mL). The organic extract was washed with brine, dried over anhydrous magnesium sulfate and concentrated under reduced pressure. The residue was subjected to a silica gel column eluting with a gradient of 10% to 50% ethyl acetate in heptane to give the title product as a white solid (40 mg, 69%). ¹ H-NMR (chloroform-d) δ: 7.24-7.42 (m, 15H), 5.37 (d, J = 8.6 Hz, 1H), 5.35 (d, J = 1.6 Hz) 1H), 4.95 (d, J = 11.3 Hz, 1H) 4.71 (d, J = 12.1 Hz, 1H), 4.56 (d, J = 11.3 Hz, 1H), 4. 45 (d, J = 12.1 Hz, 1H), 4.40 (s, 2H), 4.35-4.40 (m, 1H), 4.05 (d, J = 3.5 Hz, 1H), 3.95 (d, J = 8.6 Hz, 1H), 3.71 (d, J = 8.2 Hz, 1H), 3.61 (d, J = 8.2 Hz, 1H), 3.50 (dd , J = 10.0, 3.7 Hz, 1H), 3.45 (d, J = 8.6 Hz, 1H), 2.92 (q, J = 10.5 Hz, 2H). ¹³ C-NMR (chloroform-d) δ: 162.3, 138.0, 137.6, 137.3, 128.7, 128.5, 128.4, 128.3, 128.3, 128.2 , 128.0, 128.0, 127.8, 101.1, 82.8, 75.8, 75.0, 73.7, 73.2, 71.9, 70.2, 69.3, 54 0.0, 41.7. ¹⁹ F-NMR (chloroform-d) δ: -62.8 (s). LCMS (ES-): 2.05 min, 570.3 (MH) ^- .

N-{(1S, 2R, 3R, 4R, 5S) -2,3-bis (benzyloxy) -1-[(benzyloxy) methyl] -6,8-dioxabicyclo [3.2.1] octa -4-yl} -2,2-difluoroacetamide (1-n-7)
To a stirred mixture of difluoroacetic acid (29 mg, 0.31 mmol) in N, N-dimethylformamide (1 mL) was added 1 mL of 1,1′-carbonyldiimidazole (33 mg, 0.20 mmol) in one portion at room temperature. The solution was stirred at room temperature for 3 hours. Triethylamine (0.028 mL, 0.20 mmol) and (1S, 2R, 3R, 4R, 5S) -2,3-bis (benzyloxy) -1-((benzyloxy) methyl) -6,8-dioxabicyclo [3.2.1] Octane-4-amine (1-n-1) (47 mg, 0.10 mmol) was added in one portion at room temperature. The reaction mixture was stirred at room temperature for 16 hours and partitioned between ethyl acetate (3 mL), brine (2 mL) and water (2 mL). The organic extract was washed with brine, dried over anhydrous magnesium sulfate and concentrated. The residue was subjected to silica gel column chromatography eluting with a gradient of 10% to 50% ethyl acetate in heptane to give the title product as a white solid (32 mg, 58%). ¹ H-NMR (chloroform-d) δ: 7.14-7.47 (m, 15H), 6.02 (d, J = 8.6 Hz, 1H), 5.83 (t, J = 54.2 Hz) , 1H), 5.35 (s, 1H), 4.96 (d, J = 11.3 Hz, 1H), 4.73 (d, J = 12.5 Hz, 1H), 4.58 (d, J = 11.3 Hz, 1H), 4.45 (d, J = 12.1 Hz, 1H), 4.41 2H), 4.34-4.40 (m, 1H), 4.08 (d, J = 3.9 Hz, 1H), 3.96 (d, J = 9.0 Hz, 1H), 3.67-3.75 (m, 1H), 3.59-3.65 (m, 1H), 53 (dd, J = 9.8, 3.9 Hz, 1H), 3.46 (d, J = 8.6 Hz, 1H). ¹³ C-NMR (chloroform-d) δ: 162.6, 138.0, 137.3, 128.8, 128.5, 128.4, 128.3, 128.1, 128.0, 127.8 , 108.3 (t, J = 253.1 Hz), 72.9, 71.6, 70.2, 69.3, 53.5. ¹⁹ F-NMR (chloroform-d) δ: -126.1 (d, J = 53.1 Hz). LCMS (ES-): 2.05 min, 538.2 (M-H) ^- .

tert-butyl {(1S, 2R, 3R, 4R, 5S) -2,3-bis (benzyloxy) -1-[(benzyloxy) methyl] -6,8-dioxabicyclo [3.2.1] Oct-4-yl} carbamate (1-n-8)
(1S, 2R, 3R, 4R, 5S) -2,3-bis (benzyloxy) -1-((benzyloxy) methyl) -6,8-dioxabicyclo [3.2.1] octane-4- To a stirred mixture of amine (1-n-1) (120 mg, 0.26 mmol) and N, N-dimethylaminopyridine (DMAP) (6.4 mg, 0.052 mmol) and tetrahydrofuran (2 mL) was added di-tert-butyl. Dicarbonate (113 mg, 0.52 mmol) was added in one portion at room temperature. The reaction mixture was stirred at room temperature for 16 hours and concentrated under reduced pressure. The residue was purified on a silica gel column eluting with a gradient of 10% to 40% ethyl acetate heptane solution in to give the title product as a white solid (104 mg, 71%). ¹ H-NMR (chloroform-d) δ: 7.17-7.45 (m, 15H), 5.35 (s, 1H), 4.97 (d, J = 11.3 Hz, 1H), 4. 74 (d, J = 12.1 Hz, 1H), 4.57 (d, J = 11.3 Hz, 2H), 4.47 (d, J = 7.0 Hz, 1H), 4.39 (s, 2H) ), 4.09 (br.s, 1H), 4.01 (d, J = 3.9 Hz, 1H), 3.93 (d, J = 9.0 Hz, 1H), 3.63-3.71 (M, 1H), 3.54-3.62 (m, 1H), 3.38-3.48 (m, 2H), 1.47 (s, 9H). ¹³ C-NMR (chloroform-d) δ: 155.2, 138.2, 137.8, 137.4, 128.5, 128.4, 128.3, 128.3, 128.0, 127.9 , 127.8, 127.8, 127.7, 102.2, 82.8, 79.4, 74.8, 73.7, 73.4, 72.0, 70.0, 69.4, 54 .6, 31.9, 28.4. LCMS (AP +): 2.25min, 462.2 (M-Boc + H) ^<+> .

N-[(1S, 2R, 3R, 4R, 5S) -2,3-dihydroxy-1- (hydroxymethyl) -6,8-dioxabicyclo [3.2.1] oct-4-yl] propanamide (25)
N- (1S, 2R, 3R, 4R, 5S) -2,3-bis (benzyloxy) -1-((benzyloxy) methyl) -6,8-dioxabicyclo [3.2.1] octane- 4-yl] propanamide (1-n-5) (42 mg, 0.081 mmol), 1-methyl-1,4-cyclohexadiene (0.093 mL, 0.81 mmol), 10% Pd on activated carbon (20 mg) And a mixture of 2-propanol (2.5 mL) was stirred at 80 ° C. for 3 hours. Water (0.2 mL) was added and the entire mixture was loaded onto silica gel and dried on a rotary evaporator. This material was purified on a silica gel column eluting with a gradient of 4% to 15% methanol in dichloromethane to give the title compound as a colorless gum (12 mg, 60%). ¹ H-NMR (methanol-d ₄ ) δ: 5.21 (d, J = 1.6 Hz, 1H), 3.95 (dd, J = 10.1, 1.2 Hz, 1H), 3.92 ( d, J = 11.3 Hz, 1H), 3.87 (d, J = 1H), 3.81 (d, J = 11.3 Hz, 1H), 3.75 (d, J = 8.2 Hz, 1H) ), 3.71-3.76 (m, 2H), 3.68 (d, J = 8.2 Hz, 1H), 3.35 (s, 1H), 2.26 (q, J = 7.4 Hz) , 2H), 1.13 (t, J = 7.4 Hz, 3H). ¹³ C-NMR (methanol-d ₄ ) δ: 177.7, 102.6, 84.9, 70.4, 69.1, 69.0, 61.9, 56.0, 30.0, 10. 3 LCMS (AP +): 0.25 min, 248.2 (M + H) ^<+> .

N-[(1S, 2R, 3R, 4R, 5S) -2,3-dihydroxy-1- (hydroxymethyl) -6,8-dioxabicyclo [3.2.1] oct-4-yl] -2 , 2,2-Trifluoroacetamide (24)
N-((1S, 2R, 3R, 4R, 5S) -2,3-bis (benzyloxy) -1-((benzyloxy) methyl) -6,8-dioxabicyclo [3.2.1] octane -4-yl) -2,2,2-trifluoroacetamide (1-n-3) (20 mg, 0.036 mmol), 1-methyl-1,4-cyclohexadiene (0.093 mL, 0.81 mmol), A mixture of 10% Pd (20 mg) and 2-propanol (2.5 mL) on activated carbon was stirred at 80 ° C. for 3 hours. Water (0.2 mL) was added and the entire mixture was loaded onto silica gel and dried on a rotary evaporator. This material was purified on a silica gel column eluted with a gradient of 4% to 15% methanol in dichloromethane to give the title compound as a colorless gum (7.2 mg, 69%). ¹ H-NMR (methanol-d ₄ ) δ: 5.25 (d, J = 1.2 Hz, 1H), 4.02 (d, J = 8.6 Hz, 1H), 3.90 (d, J = 7.0 Hz, 1H), 3.88-3.95 (m, 2H), 3.82 (d, J = 11.7 Hz, 1H), 3.78 (d, J = 7.8 Hz, 1H), 3.71 (d, J = 7.8 Hz, 1H), 3.35 (s, 1H). ¹³ C-NMR (methanol-d ₄ ) δ: 159.8, 102.3, 85.5, 70.8, 69.7, 68.4, 62.2, 57.4. ¹⁹ F-NMR (chloroform-d) δ: −77.0 (s). LCMS (AP +): 0.42 min, 288.2 (M + H) ⁺ .

N-[(1S, 2R, 3R, 4R, 5S) -2,3-dihydroxy-1- (hydroxymethyl) -6,8-dioxabicyclo [3.2.1] oct-4-yl] methanesulfone Amide (26)
N-((1S, 2R, 3R, 4R, 5S) -2,3-bis (benzyloxy) -1-((benzyloxy) methyl) -6,8-dioxabicyclo [3.2.1] octane -4-yl) methanesulfonamide (1-n-4) (19 mg, 0.035 mmol), 1-methyl-1,4-cyclohexadiene (0.093 mL, 0.81 mmol), 10% Pd on activated carbon ( 20 mg) and 2-propanol (2.5 mL) were stirred at 80 ° C. for 3 hours. Water (0.2 ml) was added and the entire mixture was loaded onto silica gel and dried on a rotary evaporator. This material was purified on a silica gel column eluting with a gradient of 4% to 15% methanol in dichloromethane to give the title compound as a colorless gum (6.4 mg, 68%). ¹ H-NMR (methanol-d ₄ ) δ: 5.26 (d, J = 1.6 Hz, 1H), 3.91 (d, J = 11.3 Hz, 1H), 3.87 (d, J = 4.3 Hz, 1H), 3.80 (d, J = 11.3 Hz, 1H), 3.73 (d, J = 7.8 Hz, 1H), 3.68 (d, J = 7.8 Hz, 1H) ), 3.66-3.71 (m, 1H), 3.37 (dd, J = 9.8, 1.6 Hz, 1H), 3.35 (s, 1H), 3.04 (s, 3H) ). ¹³ C-NMR (methanol-d ₄ ) δ: 104.6, 85.1, 70.9, 69.8, 69.3, 62.0, 60.2, 41.7. LCMS (ES-): 0.15 min, 268.0 (M-H) ^- .

N-[(1S, 2R, 3R, 4R, 5S) -2,3-dihydroxy-1- (hydroxymethyl) -6,8-dioxabicyclo [3.2.1] oct-4-yl] -2 , 2-Difluoroacetamide (27)
N-{(1S, 2R, 3R, 4R, 5S) -2,3-bis (benzyloxy) in 2-propanol (1.0 mL) and tetrahydrofuran (0.5 mL) in a 5 mL microwave vial -1-[(benzyloxy) methyl] -6,8-dioxabicyclo [3.2.1] oct-4-yl} -2,2-difluoroacetamide (1-n-7) (32.0 mg, 0.059 mmol) solution was added 1-methyl-1,4-cyclohexadiene (0.2 mL, 2 mmol) followed by 10% palladium on carbon (50% wet wt / wt, 20.0 mg, 0 mmol). ) Was added. The vial was sealed and heated to 80 ° C. for 4 hours. After 4 hours, TLC (10% methanol / dichloromethane) indicated that the reaction was not complete but the desired product was formed. Additional 1-methyl-1,4-cyclohexadiene (0.2 mL mg, 2 mmol) was added and the reaction was resealed and heated to 80 ° C. overnight (18 hours). After a total of 22 hours, the reaction was diluted with methanol and filtered through a Life Sciences Acrodisc 25 mm syringe filter. The filtrate was concentrated under reduced pressure. The crude material was purified using CombiFlash Rf (RediSep 4 g silica gel column) eluting with a gradient of 0% to 20% methanol / dichloromethane to give the title compound as a solid (5.0 mg, solid 31%). Method C: LRMS operating for 3 minutes [M + Na = 292]. ¹ H-NMR (methanol-d ₄ ) δ: 6.06 (t, J = 54.2 Hz, 1H), 5.25 (s, 1H), 4.02 (d, J = 9.4 Hz, 1H) 3.92 (d, J = 11.7 Hz, 1H), 3.84-3.90 (m, 2H), 3.81 (d, J = 11.7 Hz, 1H), 3.78 (d, J = 8.2 Hz, 1H), 3.70 (d, J = 8.2 Hz, 1H).

N-[(1S, 2R, 3R, 4R, 5S) -2,3-dihydroxy-1- (hydroxymethyl) -6,8-dioxabicyclo [3.2.1] oct-4-yl] -3 , 3,3-trifluoropropanamide (28)
N-{(1S, 2R, 3R, 4R, 5S) -2,3-bis (benzyloxy) in 2-propanol (1.0 mL) and tetrahydrofuran (0.5 mL) in a 5 mL microwave vial -1-[(benzyloxy) methyl] -6,8-dioxabicyclo [3.2.1] pyridin-4-yl} -3,3,3-trifluoropropanamide (1-n-6) To the solution was added 1-methyl-1,4-cyclohexadiene (0.2 mL, 1.75 mmol) followed by 10% palladium on carbon (50% wet wt / wt, 20.0 mg, 0 mmol). . The vial was sealed and heated to 80 ° C. for 4 hours. After 4 hours, the reaction was diluted with methanol and filtered through a Life Sciences Acrodisc 25 mm syringe filter. The filtrate was concentrated under reduced pressure. The crude material was purified using CombiFlash Rf (RediSep 4 g silica gel column) eluting with a gradient of 0% to 20% methanol / dichloromethane to give the title compound as a solid (15.3 mg, 73 %). LRMS [M + 1 = 302]. ¹ H-NMR (methanol-d ₄ ) δ: 5.23 (s, 1H), 3.99 (d, J = 9.8 Hz, 1H), 3.92 (d, J = 11 Hz, 1H), 3 .87 (d, J = 4.3 Hz, 1H), 3.81 (d, J = 11.3 Hz, 1H), 3.76 (d, J = 8.2 Hz, 1H), 3.71-3. 74 (m, 1H), 3.69 (d, J = 8.2 Hz, 1H), 3.22 (qd, J = 10.7, 2.5 Hz, 2H)

N-{(1S, 2R, 3R, 4R, 5S))-2,3-bis (benzyloxy) -1-[(benzyloxy) methyl] -6,8-dioxabicyclo [3.2.1] Oct-4-yl} -N-methylacetamide (1-o-1)
N-((1S, 2R, 3R, 4R, 5S) -2,3-bis (benzyloxy) -1-((benzyloxy) methyl) -6,8-dioxabicyclo [3.2.1] octane To a stirred mixture of -4-yl) acetamide (1-n-2) (19 mg, 0.038 mmol) and N, N-dimethylformamide (1.5 mL) was added sodium hydride (60% suspension in mineral oil). Was added in one portion at room temperature and the mixture was stirred for 30 minutes. Iodomethane (16 mg, 0.11 mmol) was added in one portion at room temperature. The reaction mixture was stirred at room temperature for 16 hours and partitioned between ethyl acetate (3 mL), brine (2 mL) and water (2 mL). The organic extract was washed with brine, dried over anhydrous magnesium sulfate and concentrated under reduced pressure. The residue was purified on a silica gel column eluting with a gradient of 10% to 50% ethyl acetate in heptane to give the title product as a colorless gum (19 mg, 97%). A mixture of rotamers (3: 1). Rotamer l: ¹ H-NMR (chloroform-d) d: 7.21-7.43 (m, 15H), 5.37 (s, 1H), 4.93 (d, J = 10.9 Hz, 1H), 4.65 (d, J = 11.7 Hz, 1H), 4.54 (d, J = 11.3 Hz, 1H), 4.50 (d, J = 11.7 Hz, 1H), 4. 37-4.45 (m, 2H), 4.15 (d, J = 9.8 Hz, 1H), 4.09 (d, J = 3.5 Hz, 1H), 3.93 (d, J = 8 .6 Hz, 1H), 3.85 (dd, J = 10.1, 3.5 Hz, 1H), 3.78 (d, J = 8.2 Hz, 1H), 3.64 (d, J = 8. 2Hz, 1H), 3.45 (d, J = 8.6 Hz, 1H), 2.80 (s, 3H), 2.19 (s, 3H). Rotamer 2: ¹ H-NMR (chloroform-d) d: 7.21-7.43 (m, 15H), 5.27 (s, 1H), 5.12 (d, J = 10.9 Hz, 1H), 4.94-4.98 (m, 1H), 4.74 (d, J = 12.1 Hz, 1H), 4.56 (d, J = 11.7 Hz, 1H), 4.46 ( d, J = 11.3 Hz, 1H), 4.37-4.46 (m, 2H), 4.14-4.17 (m, 1H), 3.97 (d, J = 8.6 Hz, 1H). ), 3.82-3.89 (m, 2H), 3.79 (d, J = 8.2 Hz, 1H), 3.60-3.63 (m, 1H), 2.71 (s, 3H) ), 2.08 (s, 3H). ¹³ C-NMR (chloroform-d) d: 172.2, 138.0, 137.3, 137.2, 128.6, 128.5, 128.5, 128.4, 128.1, 128.0 , 127.9, 127.9, 127.9, 127.8, 103.2, 83.2, 75.2, 74.1, 73.8, 73.7, 73.0, 72.4, 70 .1, 69.2, 61.2, 28.0, 22.2. LCMS (AP +): 1.99 min, 518.0 (M + H) ⁺ .

N-{(1S, 2R, 3R, 4R, 5S) -2,3-bis (benzyloxy) -1-[(benzyloxy) methyl] -6,8-dioxabicyclo [3.2.1] octa -4-yl} -N-methylmethanesulfonamide (1-o-2)
N-((1S, 2R, 3R, 4R, 5S) -2,3-bis (benzyloxy) -1-((benzyloxy) methyl) -6,8-dioxabicyclo [3.2.1] octane To a stirred mixture of -4-yl) methanesulfonamide (1-n-4) (19 mg, 0.035 mmol) and N, N-dimethylformamide (1.5 mL), sodium hydride (60% suspension in mineral oil). Solution) was added in one portion at room temperature and the mixture was stirred for 30 minutes. Iodomethane (16 mg, 0.11 mmol) was added in one portion at room temperature. The reaction mixture was stirred at room temperature for 16 hours and partitioned between ethyl acetate (3 mL), brine (2 mL) and water (2 mL). The organic extract was washed with brine, dried over anhydrous magnesium sulfate and concentrated under reduced pressure. The residue was purified on a silica gel column eluting with a gradient of 10% to 50% ethyl acetate in heptane to give the title product as a colorless gum (11 mg, 56%). ¹ H-NMR (chloroform-d) δ: 7.25-7.39 (m, 15H), 5.39 (d, J = 0.8 Hz, 1H), 4.93 (d, J = 11.7 Hz) , 1H), 4.80 (d, J = 11.3 Hz, 1H), 4.58 (d, J = 11.3 Hz, 1H), 4.45 (d, J = 11.7 Hz, 1H), 4 .43 (d, J = 11.7 Hz, 1H), 4.41 (d, J = 11.7 Hz, 1H), 4.21 (d, J = 3.5 Hz, 1H), 4.18 (d, J = 10.5 Hz, 1 H), 3.98 (d, J = 8.6 Hz, 1 H), 3.87 (dd, J = 10.5, 3.9 Hz, 1 H), 3.82 (d, J = 8.6 Hz, 1H), 3.62 (d, J = 8.2 Hz, 1H), 3.45 (d, J = 9.0 Hz, 1H), 2.83 ( s, 3H), 2.68 (s, 3H). ¹³ C-NMR (chloroform-d) δ: 138.1, 137.3, 136.8, 128.7, 128.5, 128.4, 128.4, 128.1, 128.1, 128.0 , 127.8, 128.1, 104.6, 82.9, 74.9, 73.8, 73.5, 73.3, 71.6, 70.3, 69.4, 59.7, 37 .5, 29.5. LCMS (AP +): 2.08 min, 575.8 (M + Na) ⁺ .

tert-butyl {(1S, 2R, 3R, 4R, 5S) -2,3-bis (benzyloxy) -1-[(benzyloxy) methyl] -6,8-dioxabicyclo [3.2.1] Oct-4-yl} methylcarbamate (1-o-3)
Tert-Butyl {(1S, 2R, 3R, 4R, 5S) -2,3-bis (benzooxy) -1-[(benzyloxy) methyl] -6 in N, N-dimethylformamide (1.5 mL) , 8-dioxabicyclo [3.2.1] oct-4-yl} carbamate (1-n-8) (100 mg, 0.178 mmol) was added to sodium hydride (60% dispersion in mineral oil). 8.55 mg, 0.214 mmol) was added at room temperature. The reaction was stirred for 1 hour before iodomethane (0.055 mL, 0.89 mmol) was added. The reaction was stirred overnight at room temperature. After 24 hours, the reaction was quenched with water and extracted three times with ethyl acetate. The combined organic layers were washed with water, brine, dried over sodium sulfate, filtered and concentrated under reduced pressure. The crude material was purified using a CombiFlash Rf (RediSep 12 g silica gel column) eluting with a 0% to 100% ethyl acetate / dichloromethane gradient to give the title compound (78 mg, 76%). Method C: LRMS operating for 3 minutes [M + Na = 598]. ¹ H-NMR (compound is an approximately 1: 1 mixture of two rotamers).
Rotamer 1: ¹ H-NMR (methanol-d ₄ ) δ: 7.08-7.48 (m, 15H), 5.26 (s, 1H), 4.83-4.90 (m, 1H ), 4.73 (d, J = 11.7 Hz, 1H), 4.47-4.58 (m, 3H), 4.36-4.47 (m, 2H), 4.21 (d, J = 3.5 Hz, 1H), 3.98 (dd, J = 10.7, 3.3 Hz, 1H), 3.92 (dd, J = 8.2 Hz, 2H), 3.61 (d, J = 7.8 Hz, 1 H), 3.47 (dd, J = 8.6, 3.9 Hz, 1 H), 2.75 (s, 3 H), 1.42 (s, 9 H)
Rotamer 2: ¹ H-NMR (methanol-d ₄ ) δ: 7.08-7.48 (m, 15H), 5.21 (s, 1H), 4.83-4.90 (m, 1H ), 4.73 (d, J = 11.7 Hz, 1H), 4.47-4.58 (m, 3H), 4.36-4.47.
(M, 2H), 4.21 (d, J = 3.5 Hz, 1H), 3.98 (dd, J = 10.7, 3.3 Hz, 1H), 3.92 (d, J = 8. 2 Hz, 1H), 3.61 (d, J = 7.8 Hz, 1H), 3.47 (dd, J = 8.6, 3.9 Hz, 1H), 2.70 (s, 3H), 44-1.52 (m, 9H)

N-[(1S, 2R, 3R, 4R, 5S) -2,3-dihydroxy-1- (hydroxymethyl) -6,8-dioxabicyclo [3.2.1] oct-4-yl] -N -Methylacetamide (29)
N-((1S, 2R, 3R, 4R, 5S))-2,3-bis (benzyloxy) -1- (benzyloxy) methyl-6,8-dioxabicyclo [3.2.1] octa- 4-yl) -N-methylacetamide (1-o-1) (19 mg, 0.037 mmol), 1-methyl-1,4-cyclohexadiene (0.093 mL, 0.81 mmol), 10% Pd on activated carbon A mixture of (20 mg) and 2-propanol (2.5 mL) was stirred at 80 ° C. for 3 hours. Water (0.2 ml) was added and the entire mixture was loaded onto silica gel and dried on a rotary evaporator. This material was purified on a silica gel column eluting with a gradient of 4% to 15% methanol in dichloromethane to give the title compound as a colorless gum (4.3 mg, 47%). ¹ H-NMR (about 1: 1 mixture of rotamers).
Rotamer 1: ¹ H-NMR (methanol-d ₄ ) δ: 5.20 (s, 1H), 4.65 (d, J = 10.5 Hz, 1H), 4.02-4.09 (m 1H), 3.89-3.98 (m, 2H), 3.84 (s, 1H), 3.78-3.82 (m, 1H), 3.68 (s, 1H), 11 (s, 3H), 2.15 (s, 3H)
Rotamer 2: ¹ H-NMR (methanol-d ₄ ) δ: 5.37 (s, 1H), 4.02-4.09 (m, 1H), 3.89-3.98 (m, 3H) ), 3.84-3.87 (m, 1H), 3.79-3.83 (m, 1H), 3.71 (d, J = 8.2 Hz, 1H), 2.98 (s, 3H) ), 2.15 (s, 3H)
¹³ C-NMR (methanol-d ₄ ) δ: 175.4, 104.85.8, 71.3, 69.7, 66.2, 62.3, 59.1, 28.8, 22.7. LCMS (ES-): 0.41 min, 246.2 (M-H)-

N-[(1S, 2R, 3R, 4R, 5S) -2,3-dihydroxy-1- (hydroxymethyl) -6,8-dioxabicyclo [3.2.1] oct-4-yl] -N -Methylmethanesulfonamide (30)
N-((1S, 2R, 3R, 4R, 5S) -2,3-bis (benzyloxy) -1-((benzyloxy) methyl) -6,8-dioxabicyclo [3.2.1] octane -4-yl) -N-methylmethanesulfonamide (1-o-2) (11 mg, 0.020 mmol), 1-methyl-1,4-cyclohexadiene (0.093 mL, 0.81 mmol) on activated carbon A mixture of 10% Pd (20 mg) and 2-propanol (2.5 mL) was stirred at 80 ° C. for 3 hours. Water (0.2 ml) was added and the entire mixture was loaded onto silica gel and dried on a rotary evaporator. Silica gel column chromatography eluting with a gradient of methanol from 4% to 15% in methanol gave the title compound as a colorless gum (2.9 mg, 51%). ¹ H-NMR (methanol-d ₄ ) δ: 5.26 (d, J = 1.2 Hz, 1H), 4.00-4.05 (m, 1H), 3.92-3.96 (m, 1H), 3.89-3.91 (m, 1H), 3.84 (d, J = 1.2 Hz, 1H), 3.77-3.83 (m, 2H), 3.68 (d, J = 7.8 Hz, 1H), 3.35 (s, 3H), 2.93 (s, 3H). ¹³ C-NMR (methanol-d ₄ ) δ: 106.1, 85.5, 71.5, 69.7, 65.9, 62.6, 62.2, 37.8, 30.4. LCMS (ES-): 0.42 min, 282.0 (M-H) ^-

tert-Butyl [(1S, 2R, 3R, 4R, 5S) -2,3-dihydroxy-1- (hydroxymethyl) -6,8-dioxabicyclo [3.2.1] oct-4-yl] methyl Carbamate (31)
In a 5 mL microwave vial, tert-butyl {(1S, 2R, 3R, 4R, 5S) -2,3-bis (benzyloxy) in 2-propanol (1.0 mL) and tetrahydrofuran (0.5 mL). ) -1-[(Benzyloxy) methyl] -6,8-dioxabicyclo [3.2.1] oct-4-yl} methylcarbamate (1-o-3) (75 mg, 0.13 mmol) To was added 1-methyl-1,4-cyclohexadiene (0.18 mL, 1.56 mmol) followed by 10% palladium on carbon (50% wet wt / wt, 20.0 mg). The vial was sealed and heated to 80 ° C. for 4 hours. TLC after 4 hours (10% methanol / dichloromethane) indicated that the reaction was not complete, but the desired product was formed. Additional 1-methyl-1,4-cyclohexadiene (0.18 mL, 1.6 mmol) was added and the reaction was resealed and heated to 80 ° C. overnight (18 hours). After a total of 22 hours, the reaction was diluted with methanol and filtered through a Life Sciences Acrodisc 25 mm syringe filter. The filtrate was concentrated under reduced pressure. The crude material was purified using CombiFlash Rf (RediSep 4 g silica gel column) eluting with a methanol / dichloromethane gradient from 0% to 20% to give the title compound as a solid (29.0 mg, 73% ). Method C: LRMS [M + Na = 328] running for 3 minutes. The compound is an approximately 1: 1 mixture of two rotamers:
Rotamer 1: ¹ H-NMR (methanol-d ₄ ) δ :: 5.22 (br.s., 1H), 4.19 (d, J = 10.6 Hz, 1H), 4.00 (d , J = 10.6 Hz, 1H), 3.90-3.95 (m, 2H), 3.77-3.82 (m, 2H), 3.67 (d, J = 7.6 Hz, 1H) , 2.94 (s, 3H), 1.47 (s, 9H)
Rotamer 2: ¹ H-NMR (methanol-d ₄ ) δ: 5.21 (br.s., 1H), 4.05-4.10 (m, 1H), 4.00 (d, J = 10.6 Hz, 1H), 3.90-3.95 (m, 2H), 3.77-3.82 (m, 2H), 3.67 (d, J = 7.6 Hz, 1H), 94 (s, 3H), 1.47 (s, 9H)

(1S, 2R, 3R, 4R, 5S) -1- (hydroxymethyl) -4- (methylamino) -6,8-dioxabicyclo [3.2.1] octane-2,3-diol hydrochloride ( 32)
Tert-Butyl [(1S, 2R, 3R, 4R, 5S) -2,3-dihydroxy-1- (hydroxymethyl) -6,8-dioxabicyclo [3.2. To a solution of 1] oct-4-yl] methylcarbamate (31) (25.3 mg, 0.0829 mmol) was added 4.0 M hydrogen chloride in dioxane (0.518 mL, 2.07 mmol) and the reaction was added. Stir at room temperature for 48 hours. After 48 hours, the reaction was concentrated under reduced pressure. The crude material was washed with ethyl acetate (5 mL) which produced a solid that was diluted with heptane (10 mL) and concentrated under reduced pressure to give the title compound as a solid (30.0 mg, 130%). . Method C: 3-minute run LRMS [M + 1 = 206]. ¹ H-NMR (methanol-d ₄ ) δ: 5.63 (s, 1H), 3.90-3.97 (m, 3H), 3.84 (s, 2H), 3.78 (d, J = 8.2 Hz, 1H), 3.10-3.20 (m, 1H), 2.84 (s, 3H)

N-[(1S, 2R, 6R, 7R, 8S) -4,4-dimethyl-1- (15-phenyl-2,5,8,11,14-pentaoxapentadec-1-yl) -3, 5,9,11-tetraoxatricyclo [6.2.1.0-2,6-] undec-7-yl] acetamide (l-e-3)
N-[(1S, 2R, 6R, 7R, 8S) -1- (hydroxymethyl) -4,4-dimethyl-3,5,9,11-tetraoxatricyclo [3] in dichloromethane (30.0 mL). 6.2.1.0-2,6-] Undec-7-yl] acetamide (Ie-1) (1200 mg, 4.39 mmol) and 13-iodo-1-phenyl-2,5,8,11 -Tetraoxatridecane
(Synthetic Metals, 162 (23), 2163-2170, 2012, 7000 mg, 17.76 mmol) to a solution of tetrabutylammonium hydrogen sulfate (2290 mg, 6.60 mmol), followed by 12.5 M aqueous sodium hydroxide solution (30.0 mL, 380 mmol) was added. The reaction was stirred at room temperature for 64 hours. After 64 hours, the reaction was diluted with water and dichloromethane. The layers were separated and the aqueous layer was extracted twice more with dichloromethane. The combined organic layers were washed with 1N hydrochloric acid, dried over magnesium sulfate, filtered and concentrated under reduced pressure. Ethyl acetate (50 mL) was added to the resulting crude material and stirred for 30 minutes. The resulting precipitate was filtered. The filtrate was concentrated under reduced pressure. The crude material was eluted with 0% to 100% ethyl acetate / heptane gradient using CombiFlash • Rf (ISCO • RediSep • Gold • 80 g silica gel column) immediately followed by methanol / dichloromethane from 0% to 20%. Elution with a gradient gave the title compound (1267 mg, 53.5%). Method C: 1.5 minute run LRMS [M + Na = 562]. ¹ H-NMR (methanol-d ₄ ) δ: 7.13-7.45 (m, 5H), 5.22 (d, J = 1.6 Hz, 1H), 4.55 (s, 2H), 4 .30 (d, J = 5.9 Hz, 1H), 4.15 (t, J = 6.4 Hz, 1H), 3.89-3.97 (m, 2H), 3.85 (d, J = 7.8 Hz, 1H), 3.73-3.79 (m, 2H), 3.58-3.71 (m, 16H), 1.98 (s, 3H), 1.48 (s, 3H) , 1.33 (s, 3H)

N-[(1S, 2R, 3R, 4R, 5S) -2,3-dihydroxy-1- (15-phenyl} -2,5,8,11,14-pentaoxapentadec-1-yl) -6 , 8-dioxabicyclo [3.2.1] oct-4-yl] acetamide (33)
N-[(1S, 2R, 6R, 7R, 8S) -4,4-dimethyl-1- (15-phenyl) in acetic acid (4.0 mL), methanol (1.0 mL) and water (1.0 mL) -2,5,8,11,14-pentaoxapentadec-1-yl) -3,5,9,11-tetraoxatricyclo [6.2.1.0-2,6-] undec-7 A solution of -yl] acetamide (1-e-3) (60.0 mg, 0.11 mmol) was heated to 70 ° C. overnight. After 18 hours, the reaction was cooled to room temperature and concentrated under reduced pressure. The crude material was diluted with toluene and concentrated under reduced pressure. The crude material was diluted a second time with toluene and concentrated under reduced pressure. The crude material was purified using CombiFlash Rf (RediSep 4 g Gold silica gel column) eluting with a methanol / dichloromethane gradient from 0% to 20% to give the title compound as a gum (42. 5 mg, 77%). Method C: 3-minute operation LRMS [M + 1 = 500]. ¹ H-NMR (methanol-d ₄ ) δ: 7.14-7.45 (m, 5H), 5.21 (s, 1H), 4.55 (s, 2H), 3.92-4.01 (M, 2H), 3.88 (d, J = 4.3 Hz, 1H), 3.77 (d, J = 7.8 Hz, 1H), 3.70 (dd, J = 9.8, 3. 9Hz, 1H), 3.58-3.68 (m, 18H), 1.98 (s, 3H)

N-[(1S, 2R, 6R, 7R, 8S) -1- (13-azido-2,5,8,11-tetraoxatridec-1-yl) -4,4-dimethyl-3,5 9,11-tetraoxatricyclo [6.2.1.0-2,6-] undec-7-yl] acetamide (l-e-2)
N-[(1S, 2R, 6R, 7R, 8S) -1- (hydroxymethyl) -4,4-dimethyl-3,5,9,11-tetraoxalate in N, N-dimethylformamide (200 ml) To a solution of tricyclo [6.2.1.0-2.6-] undec-7-yl] acetamide (1-e-1) (10.0 g, 36.59 mmol, 1.0 eq) was added potassium hydroxide. (8.21 g, 146.37 mmol, 4 eq) was added at 5 ° C. (ice / water). After the addition, the reaction mixture was stirred at 5 ° C. for 30 minutes. Then 1-azido-2- {2- [2- (2-iodoethoxy) ethoxy] ethoxy} ethane (36.13 g, 109.78 mmol, 3.0 eq) was reacted at 5 ° C. (ice / water). Added to the mixture. The reaction mixture was stirred at 5 ° C. (ice / water) for 30 minutes, the reaction mixture was heated to 27 ° C. and stirred at 27 ° C. for 18 hours. After 18 hours, the reaction mixture was poured into ice / water and extracted three times with dichloromethane (400 mL). The combined organic layers were washed 3 times with water (400 ml), brine (500 ml), dried over sodium sulfate, filtered and concentrated to give the crude product. The crude product was purified by silica gel chromatography eluting with dichloromethane: methanol = 100: 1 to 40: 1 to the title compound (10.0 g, 57.6%) as a colorless oil. Method C: LRMS run for 3 minutes [M + 45 (formic acid) = 519]. ¹ H-NMR (methanol-d ₄ ) δ: 5.23 (d, J = 2.0 Hz, 1H), 4.31 (d, J = 5.9 Hz, 1H), 4.16 (t, J = 6.6 Hz, 1H), 3.93-3.97 (m, 1H), 3.90-3.93 (m, J = 2.0 Hz, 1H), 3.86 (d, J = 7.8 Hz) , 1H), 3.78 (d, J = 3.9 Hz, 1H), 3.75 (d, J = 1.6 Hz, 1H), 3.61-3.71 (m, 14H), 3.37 (T, J = 4.9 Hz, 2H), 1.98 (s, 3H), 1.49 (s, 3H), 1.34 (s, 3H)

N-[(1S, 2R, 3R, 4R, 5S) -1- (13-azido-2,5,8,11-tetraoxatridec-1-yl) -2,3-dihydroxy-6,8- Dioxabicyclo [3.2.1] oct-4-yl] acetamide (34)
N-[(1S, 2R, 6R, 7R, 8S) -1- (13-azido-2,5,8) in acetic acid (6.0 mL), methanol (1.45 mL) and water (1.45 mL). , 11-tetraoxatridec-1-yl) -4,4-dimethyl-3,5,9,11-tetraoxatricyclo [6.2.1.0-2,6-] undec-7-yl A solution of acetamide (l-e-2) (82.0 mg, 0.17 mmol) was heated at 70 ° C. overnight. After 18 hours, the reaction was cooled to room temperature and concentrated under reduced pressure. The crude material was diluted with toluene and concentrated under reduced pressure. The crude material was diluted a second time with toluene and concentrated under reduced pressure. The crude material was purified using CombiFlash Rf (RediSep 4 g Gold silica gel column) eluting with a methanol / dichloromethane gradient from 0% to 20% to give the title compound as an oil (43.3 mg). 58%). Method C: 3-minute operation LRMS [M-1 = 433]. ¹ H-NMR (methanol-d ₄ ) δ: 5.21 (d, J = 0.8, 1H), 3.98 (d, J = 9.8 Hz, 1H), 3.94 (d, J = 9.8 Hz, 1H), 3.89 (d, J = 4.3 Hz, 1H), 3.78 (d, J = 7.8 Hz, 1H), 3.72 (dd, J = 10.1, 4 .3 Hz, 1H), 3.61-3.69 (m, 16H), 3.38 (t, J = 4.9 Hz, 2H), 1.99 (s, 3H)

N-[(1S, 2R, 6R, 7R, 8S) -4,4-dimethyl-1- (2,5,8,11-tetraoxatetradec-13-en-1-yl) -3,5,9 , 11-Tetraoxatricyclo [6.2.1.0-2,6-] undec-7-yl] acetamide (1-e-4)
N-[(1S, 2R, 6R, 7R, 8S) -1- (hydroxymethyl) -4,4-dimethyl-3,5,9,11-tetraoxatricyclo] in dichloromethane (1.5 mL) [6.2.1.0-2,6-] Undec-7-yl] acetamide (Ie-1) (50.0 mg, 0.18 mmol) and 3- {2- [2- (2-iodo) Ethoxy) ethoxy] ethoxy} prop-1-ene
To a solution of (Organic Letters, 5 (11), 1887-1890, 2003, 139.0 mg, 0.463 mmol) was added tetrabutylammonium hydrogen sulfate (95.3 mg, 0.275 mmol), followed by 12.5 M Of aqueous sodium hydroxide (0.75 mL, 9.4 mmol) was added. The reaction was stirred overnight at room temperature. After 18 hours, the reaction was diluted with water and dichloromethane. The layers were separated and the aqueous layer was extracted twice more with dichloromethane. The combined organic layers were washed with 1N hydrochloric acid, water, dried over magnesium sulfate, filtered and concentrated under reduced pressure. The resulting crude material was diluted with ethyl acetate (5 mL) and the resulting precipitate was stirred at room temperature for 30 minutes. The precipitate was filtered and the filter cake was washed with ethyl acetate (2 × 5 mL). The filtrate was concentrated under reduced pressure. The crude material was purified using CombiFlash • Rf (ISCO • RediSep • gold • 4 g silica gel column) eluting with an ethyl acetate / heptane gradient from 0% to 100%. The column was then eluted with a methanol / dichloromethane gradient from 0% to 20% to give the title compound (13.6 mg, 17%). Method C: LRMS running for 1.5 minutes [M + Na = 468]. ¹ H-NMR (methanol-d ₄ ) δ: 5.92 (ddt, J = 16.8, 10.9, 5.7 Hz, 1H), 5.28 (dd, J = 17.4, 1.4 Hz , 1H), 5.23 (d, J = 1.6 Hz, 1H), 5.16 (dd, J = 10.3, 1.0 Hz, 1H), 4.31 (d, J = 5.9 Hz, 1H), 4.15 (t, J = 6.4 Hz, 1H), 4.02 (d, J = 5.5 Hz, 2H), 3.89-3.97 (m, 2H), 3.86 ( d, J = 7.8 Hz, 1H), 3.73-3.80 (m, 2H), 3.56-3.72 (m, 12H), 1.98 (s, 3H), 1.49 ( s,
3H), 1.34 (s, 3H).

N-[(1S, 2R, 3R, 4R, 5S) -2,3-dihydroxy-1- (2,5,8,11-tetraoxatridec-13-en-1-yl) 6,8-di Oxabicyclo [3.2.1] oct-4-yl] acetamide (35)
N-[(1S, 2R, 6R, 7R, 8S) -4,4-dimethyl-1- (2,5) in acetic acid (1.0 mL), methanol (0.25 mL) and water (0.25 mL). , 8,11-tetraoxatetradec-13-en-1-yl) -3,5,9,11-tetraoxatricyclo [6.2.1.0-2,6-] undec-7-yl A solution of acetamide (1-e-4) (13.0 mg, 0.029 mmol) was heated to 70 ° C. overnight. After 18 hours, the reaction was cooled to room temperature and concentrated under reduced pressure. The crude material was diluted with toluene and concentrated under reduced pressure. The crude material was diluted a second time with toluene and concentrated under reduced pressure. The crude material was purified using CombiFlash Rf (RediSep 4 g Gold silica gel column) eluting with a methanol / dichloromethane gradient from 0% to 20% to give the title compound (6.5 mg, 55 %). Method C: 3-minute operation LRMS [M + 1 = 406]. ¹ H-NMR (methanol-d ₄ ) δ: 5.80-6.09 (m, 1H), 5.28 (dd, J = 17.2, 1.6 Hz, 1H), 5.21 (d, J = 0.8 Hz, 1H), 5.16 (dd, J = 10.5, 1.2 Hz, 1H), 4.02 (d, J = 5.5 Hz, 2H), 3.98 (d, J = 9.8 Hz, 1 H), 3.95 (d, J = 10.1 Hz, 1 H), 3.89 (d, J = 3.9 Hz, 1 H), 3.78 (d, J = 8.2 Hz, 1H), 3.71 (dd, J = 10.0, 4.5 Hz, 1H), 3.57-3.68 (m, 14H), 1.99 (s, 3H).

N-[(1S, 2R, 6R, 7R, 8S) -4,4-dimethyl-1- (2,5,8,11-tetraoxatetradec-13-in-1-yl) -3,5 9,11-tetraoxatricyclo [6.2.1.0-2,6-] undec-7-yl] acetamide (le-5)
N-[(1S, 2R, 6R, 7R, 8S) -4,4-dimethyl-1- (15-phenyl-2,5,8,11,14-pentaoxapentadecane in dichloromethane (3 mL) 1-yl) -3,5,9,11-tetraoxatricyclo [6.2.1.0-2,6-]-undec-7-yl] acetamide (Ie-1) (100.0 mg, 0.366 mmol) and 3- {2- [2- (2-iodoethoxy) ethoxy] ethoxy} prop-1-yne
To a solution of (Synthesis, (10), 1639-1644, 2010, 425.0 mg, 1.43 mmol) was added tetrabutylammonium hydrogen sulfate (191 mg, 0.550 mmol) followed by 12.5 M sodium hydroxide. Aqueous solution (1.5 mL, 19 mmol) was added. The reaction was stirred overnight at room temperature. After 18 hours, the reaction was diluted with water and dichloromethane. The layers were separated and the aqueous layer was extracted twice more with dichloromethane. The combined organic layers were washed with 1N hydrochloric acid, water, dried over magnesium sulfate, filtered and concentrated under reduced pressure. The resulting crude material was diluted with ethyl acetate (20 mL) and the resulting precipitate was stirred at room temperature for 30 minutes. The precipitate was filtered and the filter cake was washed with ethyl acetate (2 × 15 mL). The filtrate was concentrated under reduced pressure. The crude material was purified using CombiFlash • Rf (ISCO • RediSep • gold • 12 g silica gel column) eluting with an ethyl acetate / heptane gradient from 0% to 100%. The column was then eluted with a methanol / dichloromethane gradient from 0% to 20% to give the title compound (70.0 mg, 43%). Method C: LRMS operating for 1.5 minutes [M + Na = 466]. ¹ H-NMR (methanol-d ₄ ) δ: 5.23 (d, J = 1.6 Hz, 1H), 4.31 (d, J = 5.9 Hz, 1H), 4.19 (d, J = 2.3 Hz, 2H), 4.16 (t, J = 6.4 Hz, 1H), 3.90-3.97 (m, 2H), 3.86 (d, J = 7.8 Hz, 1H), 3.74-3.79 (m, 2H), 3.60-3.72 (m, 12H), 2.85 (t, J = 2.3 Hz, 1H), 1.98 (s, 3H), 1.49 (s, 3H), 1.34 (s, 3H)

N [(1S, 2R, 3R, 4R, 5S) -2,3-dihydroxy-1- (2,5,8,11-tetraoxatetradec-13-in-1-yl) -6,8-di Oxabicyclo [3.2.1] oct-4-yl] acetamide (36)
N-[(1S, 2R, 6R, 7R, 8S) -4,4-dimethyl-1- (2,5) in acetic acid (4.0 mL), methanol (1.0 mL) and water (1.0 mL). , 8,11-tetraoxatetradec-13-in-1-yl) -3,5,9,11-tetraoxatricyclo [6.2.1.0-2,6-] undec-7-yl A solution of acetamide (l-e-5) (70.0 mg, 0.16 mmol) was heated to 70 ° C. overnight. After 18 hours, the reaction was cooled to room temperature and concentrated under reduced pressure. The crude material was diluted with toluene and concentrated under reduced pressure. The crude material was diluted a second time with toluene and concentrated under reduced pressure. The crude material was purified using CombiFlash Rf (RediSep 4g Gold silica gel column) eluting with a methanol / dichloromethane gradient from 0% to 20% to give the title compound as a gum (57 .6 mg, 90%). Method C: 3-minute operation LRMS [M + 1 = 404]. ¹ H-NMR (methanol-d ₄ ) δ: 5.22 (s, 1H), 4.19 (d, J = 1.8 Hz, 2H), 3.98 (d, J = 10.0 Hz, 1H) 3.94 (d, J = 10.0 Hz, 1H), 3.89 (d, J = 4.1 Hz, 1H), 3.78 (d, J = 8.2 Hz, 1H), 3.71 ( dd, J = 10.0, 4.1 Hz, 1H), 3.60-3.69 (m, 14H), 2.86 (s, 1H), 1.99 (s, 3H)

N-[(1S, 2R, 3R, 4R, 5S) -1- (13-amino-2,5,8,11-tetraoxatridec-1-yl) -2,3-dihydroxy-6,8- Dioxabicyclo [3.2.1] oct-4-yl] acetamide (37)
N-[(1S, 2R, 3R, 4R, 5S) -1- (13-azido-2,5,8,11-tetraoxatridec-1-yl) -2,3 in ethanol (2 mL) A solution of -dihydroxy-6,8-dioxabicyclo [3.2.1] oct-4-yl] acetamide (34) (40.0 mg, 0.092 mmol) was passed through the H cube (condition: catalyst ( 10% palladium on carbon (30 × 4 mm), flow rate: 1 mL / min, temperature: room temperature, pressure = total H ₂ ) This solution was collected after passing through the H-cube, concentrated under reduced pressure and gum The title compound was obtained as (17.2 mg, 46%) Method C: LRMS [M + 1 = 409] run for 3 minutes ¹ H-NMR (methanol-d ₄ ) δ: 5.21 (s, 1H), 3 .92-4.00 (m, 2H), 3.89 (d, = 3.9 Hz, 1H), 3.78 (d, J = 8.2 Hz, 1H), 3.69-3.748 m, 1H), 3.61-3.69 (m, 14H), 3.56 (T, J = 5.1 Hz, 2H), 2.85 (t, J = 5.1 Hz, 2H), 1.99 (s, 3H)

N-[(1S, 2R, 3R, 4R, 5S) -2,3-dihydroxy-1- (13-hydroxy-2,5,8,11-tetraoxatridec-1-yl) -6,8- Dioxabicyclo [3.2.1] oct-4-yl] acetamide (38)
N-[(1S, 2R, 3R, 4R, 5S) -2,3-dihydroxy-1- (15-phenyl-2,5,8,11,14-pentaoxapentadec-1-yl) -6, 8-Dioxabicyclo [3.2.1] oct-4-yl] acetamide (33) (43 mg, 0.086 mmol) was dissolved in methanol (2 mL) and passed through an H cube (condition: catalyst (on carbon 20% palladium hydroxide (30 × 4 mm), flow rate: 1 mL / min, temperature: 60 ° C., pressure = total H ₂ ) After passing through the H-cube, the solution is collected and concentrated under reduced pressure The title compound was obtained (32.2 mg, 91%) ¹ H-NMR (methanol-d ₄ ) δ: 5.21 (s, 1H), 3.98 (d, J = 9.4 Hz, 1H) 3.95 (d, J = 10.1 Hz, 1H), 3.89 (d, J = 4.3 Hz, 1H), 3.78 (d, J = 8.2 Hz, 1H), 3.71 (dd, J = 9.8, 4.3 Hz, 1H), 3.61-3.69 (M, 16H), 3.54-3.59 (m, 2H), 1.99 (s, 3H). ¹³ C-NMR (methanol d ₄ ) δ: 174.1, 102.6, 84.3 73.8, 72.5, 71.7, 71.7 (2), 71.6, 71.5, 71.4, 70.5, 70.2, 69.0, 62.4, 56. 4, 22.7

N-[(1S, 2R, 6R, 7R, 8S) -1- (13-hydroxy-2,5,8,11-tetraoxatridec-1-yl) -4,4-dimethyl-3,5 9,11-tetraoxatricyclo [6.2.1.0-2,6-] undec-7-yl] acetamide (l-e-6)
N-[(1S, 2R, 6R, 7R, 8S) -4,4-dimethyl-1- (15-phenyl-2,5,8,11,14-pentaoxapentadec-1-yl) -3, 5,9,11-Tetraoxatricyclo [6.2.1.0-2,6-] undec-7-yl] acetamide (l-e-3) (2.897 g, 5.37 mmol) was added to methanol ( (Condition: catalyst (10% palladium on carbon (30 × 4 mm), flow rate: 1 mL / min, temperature: 60 ° C., pressure = total H ₂ )). After passing, the solution was collected and concentrated under reduced pressure to give the title compound as a gum (2.5 g, 100%) Method C: Run LRMS for 1.5 minutes [M + 1 = 450]. ¹ H-NMR (methanol _{-d 4) δ: 5.23 (d} , J = 1 6 Hz, 1H), 4.31 (d, J = 5.9 Hz, 1H), 4.16 (t, J = 6.4 Hz, 1H), 3.89-3.97 (m, 2H), 3. 86 (d, J = 7.8 Hz, 1H), 3.74-3.80 (m, 2H), 3.60-3.71 (m, 14H), 3.53-3.59 (m, 2H) ), 1.98 (s, 3H), 1.49 (s, 3H), 1.34 (s, 3H)

N-[(1S, 2R, 6R, 7R, 8S) -4,4-dimethyl-1- (13-oxo-2,5,8,11-tetraoxatridec-1-yl) -3,5 9,11-tetraoxatricyclo [6.2.1.0-2,6-] undec-7-yl] acetamide (1-e-6a)
N-[(1S, 2R, 6R, 7R, 8S) -1- (13-hydroxy-2,5,8,11-tetraoxatridec-1-yl) -4 in dichloromethane (5.0 mL) , 4-Dimethyl-3,5,9,11-tetraoxatricyclo [6.2.1.0-2,6-]-undec-7-yl] acetamide (l-e-6) (175 mg, 0. 389 mmol) solution was added Dess-Martin reagent (354 mg, 0.584 mmol) to give a mixture. After about 30 minutes, the reaction became nearly homogeneous. After 3 hours, the reaction mixture was diluted with dichloromethane, filtered through a plug of celite and washed with dichloromethane. The filtrate was concentrated under reduced pressure. The crude material was purified using a CombiFlash Rf (RediSep 24 g Gold silica gel column) eluting with a methanol / dichloromethane gradient from 0% to 20%. The tube containing the desired product was concentrated under reduced pressure. The resulting material was diluted with dichloromethane (4 mL) and diluted with ethyl ether (10 mL) to give a white precipitate. The solution was decanted and the solid was diluted with dichloromethane (2 mL) and ethyl ether (8 mL) and decanted a second time. The decanted solution was passed through a Life Science Acrodisc 25 mm syringe filter equipped with a 0.45 μm nylon membrane. The collected solution was concentrated under reduced pressure to give the title compound as a gum (65.0 mg, 37%). Method C: 3-minute operation LRMS [M + 1 = 448]. ¹ H-NMR (chloroform-d) δ: 9.74 (s, 1H), 5.63 (d, J = 9.0 Hz, 1H), 5.34 (d, J = 1.6 Hz, 1H), 4.23 (d, J = 5.9 Hz, 1H), 4.17 (s, 2H), 4.09-4.15 (m, 1H), 4.01 (t, J = 6.2 Hz, 1H) ), 3.97 (d, J = 10.1 Hz, 1H), 3.77-3.85 (m, 3H), 3.68-3.76 (m, 5H), 3.61-3.68. (M, 7H), 2.03 (s, 3H), 1.56 (s, 3H), 1.36 (s, 3H).

1-[(1S, 2R, 3R, 4R, 5S) -4- (acetylamino) -2,3-dihydroxy-6,8-dioxabicyclo [3.2.1] oct-1-yl] -2 , 5,8,11-Tetraoxatridecane-13-oic acid (38A)
N-[(1S, 2R, 6R, 7R, 8S) -4,4-dimethyl-1- (13-oxo-2,5,8) in tetrahydrofuran / t-butanol (1.5 mL / 1.5 mL) , 11-tetraoxatridec-1-yl) -3,5,9,11-tetraoxatricyclo [6.2.1.0-2,6-] undec-7-yl] acetamide (l-e -6a) (60.0 mg, 0.13 mmol) solution was treated with 2-methyl-2-butene (1.0 mL) via a glass pipette followed by subchlorine in water (1.5 mL) Treated with a solution of sodium acid (169.4 mg, 2.01 mmol) and sodium phosphate (250.0 mg, 2.58 mmol) (monobasic and monohydrate, 250 mg, 2.58 mmol). The reaction was allowed to stir at room temperature for 24 hours. After 24 hours, the reaction mixture was poured into water and extracted with ethyl acetate (3 times). The organic layer was discarded. The aqueous layer was concentrated under reduced pressure, the resulting crude product was dissolved in methanol (10 mL) and dichloromethane (100 mL), and the resulting mixture was filtered. The filtrate was concentrated under reduced pressure. The resulting material was dissolved in methanol (5 mL) and dichloromethane (50 mL), and the resulting mixture was filtered. The filtrate was purified using CombiFlash • Rf (RediSep • 4g silica gel column) eluting with a methanol / dichloromethane gradient from 0% to 100% to give the title compound as a sodium salt as a gum (40 mg, none, 67%). LRMS [M + 1 = 424]. ¹ H-NMR (methanol-d ₄ ) δ: 5.24 (s, 1H) 4.14 (2H) 3.97 (d, J = 10.1 Hz, 2H) 3.90 (d, J = 3. 9Hz, 1H) 3.81 (d, J = 7.8Hz, 1H), 3.63-3.77 (m, 15H), 2.01 (s, 3H)

1-[(1S, 2R, 6R, 7R, 8S) -7- (acetylamino) -4,4-dimethyl-3,5,9,11-tetraoxatricyclo [6.2.1.0-2 , 6-] undec-1-yl] -2,5,8,11-tetraoxatridecan-13-ylmethanesulfonate (l-e-7)
1-[(1S, 2R, 6R, 7R, 8S) -7- (acetylamino) -4,4-dimethyl-3,5,9,11-tetraoxatricyclo [1] in dichloromethane (12.4 mL). 6.2.1.0-2,6-] Undec-1-yl] -2,5,8,11-tetraoxatridecan-13-ylmethanesulfonate (1-e-6) (1117 mg, 2. (49 mmol) was added triethylamine (1.05 mL, 7.45 mmol), cooled to 0 ° C. using an ice bath, and then methanesulfonyl chloride (0.232 mL, 2.98 mmol) was added. The reaction was slowly warmed to room temperature and stirred at room temperature for 1.5 hours. After 1.5 hours the reaction was quenched with water and extracted. The layers were separated and the aqueous layer was further extracted with dichloromethane. The combined organic layers were washed with brine, dried over magnesium sulfate, filtered and concentrated under reduced pressure to give the title compound which was carried on crude (1300.0 mg, 99.2%). ). Method C: LRMS [M + Na = 550] running for 3 minutes. ¹ H-NMR (methanol-d ₄ ) δ: 5.23 (d, J = 2.0 Hz, 1H), 4.34-4.40 (m, 2H), 4.31 (d, J = 5. 9 Hz, 1H), 4.15 (t, J = 6.4, 1H), 3.89-3.97 (m, 2H), 3.86 (d, J = 7.8 Hz, 1H), 3. 72-3.81 (m, 4H), 3.59-3.71 (m, 12H), 3.11 (s, 3H), 1.98 (s, 3H), 1.48 (s, 3H) , 1.34 (s, 3H)

S- {1 [(1S, 2R, 6R, 7R, 8S) -7- (acetylamino) -4,4-dimethyl-3,5,9,11-tetraoxatricyclo [6.2.1.0 ~ 2,6 ~] undec-1-yl] -2,5,8,11-tetraoxatridecan-13-yl} ethanethioate (1-e-8)
1-[(1S, 2R, 6R, 7R, 8S) -7- (acetylamino) -4,4-dimethyl-3,5,9,11-tetraoxa in N, N-dimethylformamide (2 mL) Tricyclo [6.2.1.0-2,6-] undec-1-yl] -2,5,8,11-tetraoxatridecan-13-ylmethanesulfonate (1-e-7) (125 To a solution of 0.0 mg, 0.237 mmol) was added potassium thioacetate (135 mg, 1.18 mmol) and the reaction was stirred at room temperature for 64 hours. After 64 hours, the reaction was diluted with water and extracted three times with ethyl acetate. The combined organic layers were washed with brine, dried over sodium sulfate, filtered and concentrated under reduced pressure. The crude material was purified using CombiFlash Rf (RediSep 4 g silica gel column) eluting with a methanol / dichloromethane gradient from 0% to 20% to give the title compound as a gum (95.2 mg, 79.2%). Method C: LRMS [M + Na = 530] running for 3 minutes. ¹ H-NMR (methanol-d ₄ ) δ: 5.23 (d, J = 1.6 Hz, 1H), 4.31 (d, J = 5.9 Hz, 1H), 4.16 (t, J = 6.4 Hz, 1H), 3.90-3.97 (m, 2H), 3.86 (d, J = 7.8 Hz, 1H), 3.74-3.79 (m, 2H), 55-3.72 (m, 14H), 3.08 (t, J = 6.6 Hz, 2H), 2.32 (s, 3H), 1.98 (s, 3H), 1.49 (s, 3H), 1.34 (s, 3H)

S {1-[(1S, 2R, 3R, 4R, 5S) -4- (acetylamino) -2,3-dihydroxy-6,8-dioxabicyclo (3.2.1) oct-1-yl] -2,5,8,11-tetraoxatridecan-13-yl} ethanethioate (39)
S- {1 [(1S, 2R, 6R, 7R, 8S) -7- (acetylamino) -4, in acetic acid (6.0 mL), methanol (1.45 mL) and water (1.45 mL). 4-Dimethyl-3,5,9,11-tetraoxatricyclo [6.2.1.0-2,6-]-undec-1-yl] -2,5,8,11-tetraoxatridecane- A solution of 13-yl} ethanethioate (1-e-8) (81.0 mg, 0.16 mmol) was heated to 70 ° C. overnight. After 18 hours, the reaction was cooled to room temperature and concentrated under reduced pressure. The crude material was diluted with toluene and concentrated under reduced pressure. The crude material was purified using a CombiFlash Rf (RediSep 4 g Gold silica gel column) eluting with a methanol / dichloromethane gradient from 0% to 20% to give the title compound as a gum ( 53.7 mg, 72%). Method C: 3-minute run LRMS [M + 1 = 468]. ¹ H-NMR (methanol-d ₄ ) δ: 5.23 (s, 1H), 4.00 (d, J = 9.8 Hz, 1H), 3.97 (d, J = 9.8 Hz, 1H ), 3.91 (d, J = 4.3 Hz, 1H), 3.80 (d, J = 7.8 Hz, 1H), 3.73 (dd, J = 10.1, 4.3 Hz, 1H) , 3.63-3.70 (m, 14H), 3.60 (t, J = 6.6 Hz, 2H), 3.10 (t, J = 2H), 2.34 (s, 3H), 2 .01 (s, 3H)

N-{(1S, 2R, 3R, 4R, 5S) -2,3-dihydroxy-1- [13- (pyridin-2-yldisulfanyl) -2,5,8,11-tetraoxatridec-1- Yl] -6,8-dioxabicyclo [3.2.1] oct-4-yl} acetamide (40)
S- {1-[(1S, 2R, 3R, 4R, 5S) -4- (acetylamino) -2,3-dihydroxy-6,8-dioxabicyclo (3.2.) In methanol (3 mL). 1) Oct-1-yl] -2,5,8,11-tetraoxatridecan-13-yl} ethanethioate (39) (50 mg, 0.11 mmol) followed by 0.5 M sodium in methanol Methoxide (1.28 mL, 0.642 mmol) was added and the reaction was stirred at room temperature for 45 minutes. After 45 minutes, acetic acid (42 mg, 0.70 mmol, 0.040 mL) was added and stirred for 10 minutes. This methanol solution was added dropwise to a stirred solution of 2,2′-disulfanediyldipyridine (35.3 mg, 0.160 mmol) in a mixture of methanol (2 mL) and acetic acid (1 mL). The reaction was stirred at room temperature for 2 hours. After 2 hours, the reaction was concentrated under reduced pressure. The crude material was purified using CombiFlash • Rf (RediSep • gold • 4 g silica gel column) eluting with a methanol / dichloromethane gradient from 0% to 20% to give the title compound (31.4 mg, 55%). . Method C: LRMS [M + Na = 557] running for 3 minutes. ¹ H-NMR (methanol-d ₄ ) δ: 8.39 (d, J = 4.3 Hz, 1H), 7.94 (d, J = 8.2 Hz, 1H), 7.83 (td, J = 7.8, 1.6 Hz, 1H), 7.22 (d, J = 6.8, 5.3 Hz, 1H), 5.21 (s, 1H), 3.92-4.00 (m, 2H) ), 3.88 (d, J = 4.3 Hz, 1H), 3.77 (d, J = 7.8 Hz, 1H), 3.71 (t, J = 6.0 Hz, 3H), 3.59 -3.67 (m, 12H), 3.52-3.58 (m, 2H), 3.02 (t, J = 6.0 Hz, 2H), 1.99 (s, 3H)

tert-Butyl {1,3-bis (prop-2-yn-1-yloxy) -2-[(prop-2-yn-1-yloxy) methyl] propan-2-yl} carbamate (1-f-3 )
For the synthesis of (1-f-3), see Journal of Organic Chemistry, 73 (14), 5602-5605, 2008.

1,3-bis (prop-2-yn-1-yloxy) -2-[(prop-2-yn-1-yloxy) methyl] propan-2-amine hydrochloride (1-p-l)
Tert-Butyl {1,3-bis (prop-2-yn-1-yloxy) -2-[(prop-2-yn-1-yloxy) methyl] propan-2-yl} in dichloromethane (45 mL) To a solution of carbamate (1-f-3) (3000 mg, 8.945 mmol) was added 4.0 M hydrogen chloride in dioxane (20 mL, 89.4 mmol) and the reaction was stirred at room temperature for 18 hours. After 18 hours, the reaction was concentrated under reduced pressure to give an oil. To this crude mixture was added ethyl acetate (20 mL) and the resulting mixture was stirred. Heptane (20 mL) was added and the mixture was stirred at room temperature for 2 hours. This material was filtered and the filter cake was washed with ethyl acetate and evacuated to dryness for 2 hours to give the title compound (2140 mg, 88%). ¹ H-NMR (methanol-d ₄ ) δ: 4.25 (s, 6H), 3.72 (s, 6H), 2.97 (s, 3H).

Benzyl [6-({1,3-bis (prop-2-yn-1-yloxy) -2-[(prop-2-yn-1-yloxy) methyl] propan-2-yl} amino) -6 Oxohexyl] carbamate (1-q-1)
6-{[(Benzyloxy) carbonyl] amino} hexanoic acid in N, N-dimethylformamide (4 mL) and tetrahydrofuran (20.0 mL)
To a solution of (2910 mg, 11.0 mmol) was added N- (3-dimethylaminopropyl) -N′-ethylcarbodiimide hydrochloride (1-p-1) (2150 mg, 11.0 mmol) and 1-hydroxybenzotriazole (1480 mg). 11.0 mmol) was added and the reaction was stirred at room temperature for 1 hour during which time the reaction became homogeneous. 1,3-bis (prop-2-yn-1-yloxy) -2-[(prop-2-yn-1-yloxy) methyl] propan-2-amine hydrochloride (2130 mg, 7.84 mmol) undiluted To the stirred solution was added in one portion followed by N, N-diisopropylethylamine (5.46 mL, 31.4 mmol) and the reaction was heated to 60 ° C. for 24 hours. The reaction was cooled to room temperature and stirred for 24 hours. The reaction was quenched with water (150 mL) and extracted with ethyl acetate. The aqueous layer was further washed with ethyl acetate. The combined organic layers were washed with brine, dried over sodium sulfate, filtered and concentrated under reduced pressure. Purification of the crude material using a CombiFlash • Rf (RediSep • 80 g silica gel column) eluting with a 0% to 100% ethyl acetate / heptane gradient gave the title compound as an oil that was allowed to stand. Then it solidified (3250 mg, 86%). Method C: MassLynx \ Acid_3.0 Min. olp-LRMS [M + 1 = 483]. ¹ H-NMR (methanol-d ₄ ) δ: 7.10-7.46 (m, 5H), 5.06 (s, 2H), 4.14 (d, J = 2.0 Hz, 6H), 3 .79 (s, 6H), 3.03-3.20 (m, 2H), 2.83 (t, J = 2.1 Hz, 3H), 2.18 (t, J = 7.2 Hz, 2H) , 1.59 (quin, J = 7.3 Hz, 2H), 1.44-1.54 (m, 2H), 1.28-1.40 (m, 2H)

6- (Pyridin-2-yldisulfanyl) hexanoic acid (1-r-1)
2,2′-Disulfanediyldipyridine in a mixture of ethanol (12.0 mL) and acetic acid (0.291 mL)
A solution of (1490 mg, 6.75 mmol) was stirred under nitrogen followed by 6-sulfanylhexanoic acid in ethyl acetate (6.0 mL).
(1000.0 mg, 6.75 mmol) was added dropwise. The reaction was stirred at room temperature for 2 hours. After 2 hours, the reaction was concentrated under reduced pressure. The crude material was purified using a CombiFlash • Rf (RediSep • gold • 40 g silica gel column) eluting with a 0% to 100% ethyl acetate (2% acetic acid modifier) / heptane gradient to give the crude title Compound (1170 mg) was obtained. The crude material was purified again using CombiFlash Rf (RediSep gold 40 g silica gel column) eluting with a 0% to 100% ethyl acetate (2% acetic acid modifier) / heptane gradient to give the title compound. Obtained as an oil (544 mg, 31%).

1-{[6- (Pyridin-2-yldisulfanyl) hexanoyl] oxy} pyrrolidine-2,5-dione (1-s-1)

To a solution of 6- (pyridin-2-yldisulfanyl) hexanoic acid (1-r-1) (705 mg, 2.2 mmol) in N, N-dimethylformamide (4 mL) was added N-hydroxysuccinimide (306 mg, 2. 66 mmol) was added, followed by N- (3-dimethylaminopropyl) -N′-ethylcarbodiimide hydrochloride (520 mg, 2.66 mmol). The reaction was stirred overnight at room temperature. The next morning, the reaction was quenched with water and extracted three times with dichloromethane. The combined organic layers were washed with saturated sodium bicarbonate, brine, dried over sodium sulfate, filtered and concentrated under reduced pressure. The crude material was purified using a CombiFlash Rf (RediSep 40 g gold column) eluting with a 0% to 100% ethyl acetate / heptane gradient to give the title compound (364 mg, 47%). Method C: LRMS [M + 1 = 355] running for 1.5 minutes. ¹ H-NMR (methanol-d ₄ ) δ: 8.39 (d, J = 4.7 Hz, 1H), 7.85-7.90 (m, 1H), 7.77-7.84 (m, 1H), 7.21 (dd, J = 6.6, 5.5 Hz, 1H), 2.77-2.90 (m, 6H), 2.61 (t, J = 7.2 Hz, 2H), 1.63-1.83 (m, 4H), 1.46-1.59 (m, 2H)

N-{(1,3-bis (prop-2-yn-1-yloxy) -2-[(prop-2-yn-1-yloxy) methyl] propan-2-yl} -6- (pyridine-2 -Ildisulfanyl) hexanamide (1-t-1)
1,3-bis (prop-2-yn-1-yloxy) -2-[(prop-2-yn-1-yloxy) methyl] propan-2-in N, N-dimethylformamide (2.0 mL) To a solution of amine hydrochloride (1-p-1) (100.0 mg, 0.324 mmol) was added N, N-diisopropylethylamine (0.339 mL, 1.95 mmol), and the mixture was stirred for 10 minutes, then 1-{[ 6- (Pyridin-2-yldisulfanyl) hexanoyl] oxy} pyrrolidine-2,5-dione (1-s-1) (138 mg, 0.389 mmol) was added in one portion and the reaction was then added to 60 ° C. for 16 hours. Heated. After 16 hours, the reaction was diluted with water and extracted three times with ethyl acetate. The combined organic layers were washed with water, brine, dried over sodium sulfate, filtered and concentrated under reduced pressure. The crude material was purified using a CombiFlash • Rf (RediSep • 12 g silica gel column) eluting with a 0% to 100% ethyl acetate / heptane gradient to give the title compound as a gum (66 .7 mg, 43%). Method C: LRMS [M + Na = 497] running for 1.5 minutes. ¹ H-NMR (methanol-d ₄ ) δ: 8.39 (d, J = 4.7 Hz, 1H), 7.85-7.90 (m, 1H), 7.78-7.84 (m, 1H), 7.19-7.25 (m, 1H), 4.06-4.23 (m, 6H), 3.72-3.84 (m, 6H), 2.78-2.87 ( m, 5H), 2.12-2.20 (m, 2H), 1.71 (quin, J = 7.3 Hz, 2H), 1.57 (quin, J = 7.3 Hz, 2H), 36-1.50 (m, 2H)

1-{[4- (Benzyloxy) butanoyl] oxy} pyrrolidine-2,5-dione (1-u-1)
To a solution of 4- (benzyloxy) butanoic acid (1000 mg, 3.77 mmol) in N, N-dimethylformamide (7.54 mL) was added N-hydroxysuccinimide (521 mg, 4.52 mmol) followed by N- (3-Dimethylaminopropyl) -N′-ethylcarbodiimide hydrochloride (885 mg, 4.52 mmol) was added. The reaction was stirred overnight at room temperature. The next morning, the reaction was quenched with water and extracted three times with dichloromethane. The combined organic layers were washed with saturated sodium bicarbonate, brine, dried over sodium sulfate, filtered and concentrated under reduced pressure. The crude material was eluted using a CombiFlash • Rf (RediSep • 40 g • gold column) with an ethyl acetate / heptane gradient from 0% to 100% to give the title compound (1098 mg, 100%). Method C: LRMS [M + Na = 314] running for 1.5 minutes. ¹ H-NMR (methanol-d ₄ ) δ: 7.11-7.50 (m, 5H), 4.51 (s, 2H), 3.56 (t, J = 6.0 Hz, 2H),
2.81 (s, 4H), 2.73 (t, J = 7.2 Hz, 2H), 1.99 (quin, J = 6.6 Hz, 2H)

4- (Benzyloxy) -N- {1,3-bis (prop-2-yn-1-yloxy) -2-[(prop-2-yn-1-yloxy) methyl] propan-2-yl] butanamide (1-v-1)
1,3-bis (prop-2-yn-1-yloxy) -2-[(prop-2-yn-1-yloxy) methyl] propan-2-amine tris in N, N-dimethylformamide (5 mL) To a solution of fluoroacetic acid (1-p-1) (750.0 mg, 1.62 mmol) was added N, N-diisopropylethylamine (1.69 mL, 9.71 mmol) and stirred for 10 minutes, then N, N -A solution of 1-{[4- (benzyloxy) butanoyl] oxy} pyrrolidine-2,5-dione (1-u-1) (566 mg, 1.94 mmol) in dimethylformamide (1 mL) was added and the reaction The product was heated to 60 ° C. for 72 hours. After 72 hours, the reaction was diluted with water and extracted three times with ethyl acetate. The combined organic layers were washed with water, brine, dried over sodium sulfate, filtered and concentrated under reduced pressure. The crude material was purified using CombiFlash Rf (RediSep 24 g silica gel column) eluting with a 0% to 100% ethyl acetate / heptane gradient to give the title compound as a gum (495 mg). , None, 74%). Method C: 1.5 minute run LRMS [M + 1 = 412]. ¹ H-NMR (methanol-d ₄ ) δ: 7.21-7.41 (m, 5H), 4.51 (s, 2H), 4.12 (d, J = 2.3 Hz, 6H), 3 .78 (s, 6H), 3.51 (t, J = 6.2 Hz, 2H), 2.28 (t, J = 7.2 Hz, 2H), 1.79-1.94 (m, 2H)

tert-butyl (1,3-bis [(1- {1-[(1S, 2R, 6R, 7R, 8S) -7- (acetylamino) -4,4-dimethyl-3,5,9,11- Tetraoxatricyclo [6.2.1.0-2,6-] undec-1-yl] -2,5,8,11-tetraoxatridecan-13-yl} -1H-1,2,3 -Triazol-4-yl) methoxy] -2-{[(1- {1-[(1S, 2R, 6R, 7R, 8S) -7- (acetylamino) -4,4-dimethyl-3,5 9,11-tetraoxatricyclo [6.2.1.0-2,6-] undec-1-yl] -2,5,8,11-tetraoxatridecan-13-yl} -1H-1 , 2,3-Triazol-4-yl) methoxy] methyl} propan-2-yl) carbamate (1-w 1)
To a 50 mL round bottom flask equipped with a stir bar, add tert-butyl {1,3-bis (prop-2-yn-1-yloxy) -2-[(prop-2-yn-1-yloxy) methyl) propane. -2-yl} carbamate (1-f-3) (305.0 mg, 0.909 mmol) was charged and N-[(1S, 2R, 6R, 7R, 8S) -1 in t-butanol (12 mL) -(13-azido-2,5,8,11-tetraoxatridec-1-yl) -4,4-dimethyl-3,5,9,11-tetraoxatricyclo [6.2.1.0 ~ 2,6 ~] Undec-7-yl] acetamide (1-e-2) (1433.0 mg, 3.020 mmol) was added followed by water (5 mL) followed by sodium ascorbate ( 1840mg, 9.09mmo ) Was added neat and the reaction was purged with nitrogen for 10 minutes. Copper (II) sulfate (147 mg, 0.909 mmol) was added to 1 mL of water (deionized) and stirred at room temperature for 24 hours. After 24 hours, the reaction mixture was added to saturated ammonium chloride (30 mL) and concentrated ammonium hydroxide (3 mL) to quench the reaction and extracted three times with dichloromethane (20 mL). The combined organic layers were dried over magnesium sulfate, filtered and concentrated under reduced pressure. Purification of the crude material using a CombiFlash Rf (RediSep 80 g Gold silica gel column) eluting with a methanol / dichloromethane gradient from 0% to 20% gave the title compound as a white foam. (789.0 mg, none, 49.3%). Method C: 1.5 minute run LRMS [M + 45-1 = 1804]. ¹ H-NMR (methanol-d ₄ ) δ: 8.00 (s, 3H), 5.23 (d, J = 1.6 Hz, 3H), 4.51 to 4.63 (m, 12H), 4 .29 (d, J = 5.9 Hz, 3H), 4.16 (t, J = 6.4 Hz, 3H), 3.87-3.96 (m, 12H), 3.84 (d, J = 7.8 Hz, 3H), 3.73-3.80 (m, 6H), 3.64-3.72 (m, 12H), 3.54-3.63 (m, 30H), 1.98 ( s, 9H), 1.48 (s, 9H), 1.40 (s, 9H), 1.33 (s, 9H)

N- (1,3-bis [(1- {1-[(1S, 2R, 6R, 7R, 8S) -7- (acetylamino) -4,4-dimethyl-3,5,9,11-tetra Oxatricyclo [6.2.1.0-2,6-] Undec-1-yl] -2,5,8,11-tetraoxatridecan-13-yl} -1H-1,2,3- Triazol-4-yl) methoxy] -2-{[(1- {1-[(1S, 2R, 6R, 7R, 8S) -7- (acetylamino) -4,4-dimethyl-3,5,9 , 11-tetraoxatricyclo [6.2.1.0-2,6-] undec-1-yl] -2,5,8,11-tetraoxatridecan-13-yl} -1H-1, 2,3-triazol-4-yl) methoxy] methyl} propan-2-yl) -6- (pyridin-2-yldisul Aniru) hexanamide (1-x-1)
N- {1,3-bis (prop-2-yn-1-yloxy) -2-[(prop-2-yn-1-yloxy) methyl] propan-2-yl in t-butanol (2 mL) } -6- (Pyridin-2-yldisulfanyl) hexanamide (1-t-1) (66.0 mg, 0.14 mmol) and N-[(1S, 2R, 6R, 7R, 8S) -1- (13 -Azido-2,5,8,11-tetraoxatridec-1-yl) -4,4-dimethyl-3,5,9,11-tetraoxatricyclo [6.2.1.0-2 To a solution of 6-] undec-7-yl] acetamide (1-e-2) (219 mg, 0.459 mmol) was added water (0.5 mL, deionized water). Sodium ascorbate (84.3 mg, 0.417 mmol) was added as a solid and the reaction mixture was purged with nitrogen for 5 minutes before copper (II) sulfate (6.73 mg in water (0.5 mL, deionized water)). , 0.0417 mmol) was added and stirred at room temperature for 24 hours. The reaction mixture was quenched by adding saturated ammonium chloride (20 mL) and concentrated ammonium hydroxide (2 mL) and extracted three times with dichloromethane (15 mL). The combined organic layers were washed with brine, dried over magnesium sulfate, filtered and concentrated under reduced pressure. The crude material was purified using CombiFlash Rf (RediSep 12 g Gold silica gel column) eluting with a methanol / dichloromethane gradient from 0% to 20% to give impure title compound (105.0 mg, none 40%). The crude product (105.0 mg) was purified again using a CombiFlash® Rf (RediSep 4 g Gold silica gel column) eluting with a methanol / dichloromethane gradient from 0% to 20% to give the title compound as a gum (94.5 mg, 36%). Method C: MassLynx \ Acid_3.0 Min. olp-LRMS [M + Na = 11921]. ¹ H-NMR (methanol-d ₄ ) δ: 8.38 (d, J = 4.7 Hz, 1H), 7.97 (s, 3H), 7.82-7.88 (m, 1H), 7 .78-7.81 (m, 1H), 7.20 (t, J = 5.9 Hz, 1H), 5.23 (s, 3H), 4.52-4.62 (m, 12H), 4 .29 (d, J = 5.9 Hz, 3H), 4.15 (t, J = 6.4 Hz, 3H), 3.86-3.96 (m, 12H), 3.81-3.85 ( m, 3H), 3.72-3.80 (m, 12H), 3.54-3.71 (m, 36H), 2.79 (t, J = 7.2 Hz, 2H), 2.16 ( t, J = 7.2 Hz, 2H), 1.98 (s, 9H), 1.64-1.73 (m, 2H), 1.50-1.57 (m, 2H), 1.48 ( s, 9H), 1.42 (d, J = 6.6 Hz, 2H), 1.32 (s, 9H)

Benzyl {6-[(1,3-bis [(1- {1-[(1S, 2R, 6R, 7R, 8S) -7- (acetylamino) -4,4-dimethyl-3,5,9, 11-tetraoxatricyclo [6.2.1.0-2,6-] undec-1-yl] -2,5,8,11-tetraoxatridecan-13-yl} -1H-1,2 , 3-Triazol-4-yl) methoxy] -2-{[(1- {1-[(1S, 2R, 6R, 7R, 8S) -7- (acetylamino) -4,4-dimethyl-3, 5,9,11-tetraoxatricyclo [6.2.1.0-2,6-] undec-1-yl] -2,5,8,11-tetraoxatridecan-13-yl} -1H -1,2,3-triazol-4-yl) methoxy] methyl} propan-2-yl) amino] -6-oxo Hexyl} carbamate (1-y-1)
To a 250 mL round bottom flask equipped with a stir bar, add benzyl [6-({1,3-bis (prop-2-yn-1-yloxy) -2-[(prop-2-yn-1-yl) methyl]]. Propan-2-yl} amino) -6-oxohexyl] carbamate (1-q-1) (880.0 mg, 1.82 mmol) was charged and N-[(1S, 2R in t-butanol (26 mL). , 6R, 7R, 8S) -1- (13-azido-2,5,8,11-tetraoxatridec-1-yl) -4,4-dimethyl-3,5,9,11-tetraoxatri Add cyclo [6.2.1.0-2,6-] undec-7-yl] acetamide (1-e-2) (3075.0 mg, 6.8 mmol) followed by water (12 mL). Followed by sodium ascorbate (3690 mg, 18.2 mmol) was added undiluted and the reaction was purged with nitrogen for 10 minutes. Copper (II) sulfate (294 mg, 1.82 mmol) was added in 1 mL of water, and the mixture was stirred at room temperature for 24 hours. After 24 hours, the reaction mixture was added to saturated ammonium chloride (50 mL) and ammonium hydroxide (5 mL) to quench the reaction and extracted three times with dichloromethane (45 mL). The combined organic layers were dried over magnesium sulfate, filtered and concentrated under reduced pressure. The crude material was purified using CombiFlash Rf (RediSep 80 g Gold silica gel column) eluting with a methanol / dichloromethane gradient from 0% to 20% to give the title compound as a solid impure title Obtained as a compound (1890.0 mg, 54.4%). This crude product (1270.0 mg, 36.5%) was eluted using a CombiFlash Rf (RediSep 80 g Gold silica gel column) with a methanol / dichloromethane gradient from 0% to 20%. 0.0 mg, 17.5%). Total yield of the title compound 2.497 g (72%). Method C: 3-minute operation LRMS [M + 1 = 1907]. ¹ H-NMR (methanol-d ₄ ) δ: 7.9 (s, 3H), 7.21-7.47 (m, 5H), 5.25 (d, J = 1.6 Hz, 3H), 5 .07 (s, 2H), 4.53-4.62 (m, 12H), 4.31 (d, J = 5.9 Hz, 3H), 4.18 (t, J = 6.4 Hz, 3H) 3.88-3.98 (m, 12H), 3.85 (d, J = 7.8 Hz, 3H), 3.74-3.81 (m, 12H), 3.53-3.71 ( m, 36H), 3.10 (q, J = 6.2 Hz, 2H), 2.18 (t, J = 7.2 Hz, 2H), 2.00 (s, 9H), 1.53-1. 65 (m, 2H), 1.50 (s, 11H), 1.34 (s, 11H)

N- (1,3-bis [(1- {1-[(1S, 2R, 3R, 4R, 5S) -4- (acetylamino) -2,3-dihydroxy-6,8-dioxabicyclo [3 2.1] oct-1-yl] -2,5,8,11-tetraoxatridecan-13-yl} -1H-1,2,3-triazol-4-yl) methoxy] -2- { [(1- {1-[(1S, 2R, 3R, 4R, 5S) -4- (acetylamino) -2,3-dihydroxy-6,8-dioxabicyclo [3.2.1] octa-1 -Yl] -2,5,8,11-tetraoxatridecan-13-yl} -1H-1,2,3-triazol-4-yl) methoxy] methyl) propan-2-yl) -6- ( Pyridin-2-yldisulfanyl) hexanamide (41)
N- (1,3-bis [(1- {1-[(1S, 2R, 6R, 7R, 8S) -7- (acetyl) in acetic acid (4 mL), methanol (1 mL) and water 1.0 mL) Amino) -4,4-dimethyl-3,5,9,11-tetraoxatricyclo [6.2.1.0-2,6-]-undec-1-yl] -2,5,8,11- Tetraoxatridecan-13-yl} -1H-1,2,3-triazol-4-yl) methoxy] -2-{[(1- {1-[(1S, 2R, 6R, 7R, 8S)- 7- (acetylamino) -4,4-dimethyl-3,5,9,11-tetraoxatricyclo [6.2.1.0-2,6-]-undec-1-yl] -2,5, 8,11-tetraoxatridecan-13-yl} -1H-1,2,3-triazol-4-yl) methoxy] Chill} propan-2-yl) -6- (pyridin-2-yldisulfanyl) hexanamide (I-x-l) (94.5mg, a solution of 0.0498Mmol), it was heated for 64 hours to 70 ° C.. After 64 hours, the reaction was cooled to room temperature and concentrated under reduced pressure. The crude material was diluted with toluene and concentrated under reduced pressure. The crude material was diluted a second time with toluene and concentrated under reduced pressure to give the impure title compound as a gum (85.3 mg). The crude material was purified by reverse phase chromatography using the following conditions to give the title compound as a gum (47.6 mg, 53.8%).

Purification conditions:
The residue was dissolved in dimethyl sulfoxide (1 mL) and purified by reverse phase HPLC. Column: Waters Sunfire C18 19x100, 5u. Mobile phase A: 0.05% TFA in water (v / v). Mobile phase B: 0.05% TFA in acetonitrile (v / v). Gradient: 80% H ₂ O / 20.0% acetonitrile in 8.5 minutes to 65% H ₂ O / 35% acetonitrile in a straight line, 0% H ₂ O / 100% MeCN to 9.0 minutes, 9. Maintain 0% H ₂ O / 100% acetonitrile from 0 to 10.0 minutes. Flow rate: 25 mL / min. 47.6 mg of the title compound were obtained as a gum (retention time 2.87, observed mass = 890.4376).

QC condition:
Column: Waters Atlantis dC18 4.6 × 50, 5u. Mobile phase A: 0.05% TFA in water (v / v). Mobile phase B: 0.05% TFA in acetonitrile (v / v). 3.75 minutes 95.0% H ₂ O / 50% acetonitrile to 50% H ₂ O / 50% acetonitrile with a linear, to 5% H ₂ O / 95% acetonitrile to 4.0 minutes, 4.0 Maintain 5% H ₂ O / 95% acetonitrile until 5.0 minutes. Flow rate: 2 mL / min. Retention time 2.87, observed mass = 890.4376. Method C: LRMS [1 / 2M = 889] running for 3 minutes. ¹ H-NMR (methanol-d ₄ ) δ: 8.41 (d, J = 4.7 Hz, 1H), 7.99 (s, 3H), 7.84-7.91 (m, 2H), 7 .26 (t, J = 5.9 Hz, 1H), 5.21 (s, 3H), 4.58 (t, J = 5.0 Hz, 6H), 4.56 (s, 6H), 3.95 (T, J = 8.8 Hz, 6H), 3.89 (t, J = 5.0 Hz, 6H), 3.86-3.88 (m, 3H), 3.74-3.78 (m, 9H), 3.71 (dd, J = 9.4, 4.1 Hz, 3H), 3.54-3.67 (m, 42H), 2.80 (t, J = 7.0 Hz, 2H), 2.16 (t, J = 7.3 Hz, 2H), 1.99 (s, 9H), 1.68 (quin, J = 7.3 Hz, 2H), 1.50-1.57 (m, 2 ), 1.35-1.44 (m, 2H)

N-[(1S, 2R, 3R, 4R, 5S) -1- (13- {4-[(3-[(1- {1-[(1S, 2R, 3R, 4R, 5S) -4- ( Acetylamino) -2,3-dihydroxy-6,8-dioxabicyclo [3.2.1] oct-1-yl] -2,5,8,11-tetraoxatridecan-13-yl} -1H -1,2,3-triazol-4-yl) methoxy] -2-{[(1- {1-[(1S, 2R, 3R, 4R, 5S) -4- (acetylamino) -2,3- Dihydroxy-6,8-dioxabicyclo [3.2.1] oct-1-yl] -2,5,8,11-tetraoxatridecan-13-yl} -1H-1,2,3-triazole -4-yl} methoxy) methyl} -2-aminopropoxy) methyl] -1H-1,2,3-triazo Ru-1-yl} -2,5,8,11-tetraoxatridec-1-yl) -2,3-dihydroxy-6,8-dioxabicyclo [3.2.1] oct-4-yl Acetamide hydrochloride (42).
Tert in acetic acid (8.0 mL), methanol (2.0 mL) and water (2.0 mL)
-Butyl (1,3-bis [(1- {1-[(1S, 2R, 6R, 7R, 8S) -7- (acetylamino) -4,4-dimethyl-3,5,9,11-tetra Oxatricyclo [6.2.1.0-2,6-] Undec-1-yl] -2,5,8,11-tetraoxatridecan-13-yl} -1H-1,2,3- Triazol-4-yl) methoxy] -2-{[(1- {1-[(1S, 2R, 6R, 7R, 8S) -7- (acetylamino) -4,4-dimethyl-3,5,9 , 11-tetraoxatricyclo [6.2.1.0-2,6-] undec-1-yl] -2,5,8,11-tetraoxatridecan-13-yl} -1H-1, 2,3-triazol-4-yl) methoxy] methyl} propan-2-yl) carbamate (1-w-1) ( 10 mg, the solution of 0.119 mmol), and heated overnight 70 ° C.. After 18 hours, the reaction was cooled to room temperature and concentrated under reduced pressure. The crude material was diluted with toluene and methanol and concentrated under reduced pressure. The crude material was diluted a second time with toluene and concentrated under reduced pressure. The crude material was diluted with dichloromethane (10 mL) and methanol (4 mL) and to this was added 4.0 M hydrogen chloride in dioxane (2.0 mL, 8 mmol). The reaction mixture was stirred at room temperature overnight. After 18 hours, the reaction was concentrated under reduced pressure. The crude material was diluted with ethyl acetate (1 mL), to which heptane (10 mL) was added and concentrated under reduced pressure. This material was then placed under high vacuum for 18 hours to give the title compound as a solid (198.8 mg, 106%). Method C: LRMS [M + Na = 1561] running for 3 minutes. ¹ H-NMR (methanol-d ₄ ) δ: 8.13-8.21 (m, 3H), 5.22 (s, 3H), 4.71 (s, 9H), 4.65 (d, J = 4.7 Hz, 6H), 3.92-4.00 (m, 12H), 3.90 (d, J = 4.3 Hz, 3H), 3.58-3.80 (m, 51H), 2 .02 (s, 9H)

6-azido-N- (1,3-bis [1- {1-[(1S, 2R, 3R, 4R, 5S) -4- (acetylamino) -2,3-dihydroxy-6,8-dioxa Bicyclo [3.2.1] oct-1-yl] -2,5,8,11-tetraoxatridecan-13-yl} -1H-1,2,3-triazol-4-yl) methoxy] 2 -{[(1- {1-[(1S, 2R, 3R, 4R, 5S) -4- (acetylamino) -2,3-dihydroxy-6,8-dioxabicyclo [3.2.1] octa -1-yl] -2,5,8,11-tetraoxatridecan-13-yl} -1H-1,2,3-triazol-4-yl) methoxy] methyl} propan-2-yl) hexanamide (43)
In N, N-dimethylformamide (0.5 mL), N-[(1S, 2R, 3R, 4R, 5S) -1- (13- {4-[(3-[(1- {1-[( 1S, 2R, 3R, 4R, 5S) -4- (acetylamino) -2,3-dihydroxy-6,8-dioxabicyclo [3.2.1] oct-1-yl] -2,5,8 , 11-tetraoxatridecan-13-yl} -1H-1,2,3-triazol-4-yl) methoxy] -2-{[(1- {1-[(1S, 2R, 3R, 4R, 5S) -4- (acetylamino) -2,3-dihydroxy-6,8-dioxabicyclo [3.2.1] oct-1-yl] -2,5,8,11-tetraoxatridecane- 13-yl} -1H-1,2,3-triazol-4-yl} methoxy) methyl} -2-amino Ropoxy) methyl] -1H-1,2,3-triazol-1-yl} -2,5,8,11-tetraoxatridec-1-yl) -2,3-dihydroxy-6,8-dioxa To a solution of bicyclo [3.2.1] oct-4-yl] acetamide hydrochloride (42) (25 mg, 0.016 mmol) was added N, N-diisopropylethylamine (0.0111 mL, 0.0635 mmol), After stirring for 10 minutes, undiluted 1-[(6-azidohexanoyl) oxy] pyrrolidine-2,5-dione
(See PCT International Application 2011039511, March 24, 0111, 6.05 mg, 0.0238 mmol) was added and the reaction was stirred for 18 hours at room temperature. The reaction was then heated to 60 ° C. for 32 hours. After 32 hours, the reaction was concentrated under reduced pressure. The crude material was diluted with dimethyl sulfoxide (1 mL) and passed through a syringe filter, and the crude material was purified by reverse phase chromatography using the conditions shown below to give the title compound as a gum (6.2 mg). 23%).

Purification conditions:
The residue was dissolved in dimethyl sulfoxide (1 mL) and purified by reverse phase HPLC. Column: Waters Sunfire C18 19x100, 5u. Mobile phase A: 0.05% TFA in water (v / v). Mobile phase B: 0.05% TFA in acetonitrile (v / v). Gradient: 10.5 minutes 90.0% H ₂ O / 10.0% acetonitrile to 70% H ₂ O / 30% acetonitrile in a straight line, a straight line from 70% H ₂ O / 30% acetonitrile in 0.5 minutes To 0% H ₂ O / 100% MeCN at 0% H ₂ O / 100% acetonitrile from 11.0 to 12.0 minutes. Flow rate: 25 mL / min.

QC condition:
Column: Waters Atlantis dC18 4.6 × 50, 5u. Mobile phase A: 0.05% TFA in water (v / v). MobCe phase B: 0.05% TFA in acetonitrile (v / v). From 95.0% H ₂ O / 5.0% acetonitrile in 4.0 minutes to 5% H ₂ O / 95% acetonitrile in a straight line 5% H ₂ O / 95% acetonitrile from 4.0 to 5.0 minutes Maintain. Flow rate: 2 mL / min. Retention time 1.77, observed mass = 839.7097. Method C: 3 minute run LRMS [M + 1 = 1678]. ¹ H-NMR (methanol-d ₄ ) δ: 8.00 (s, 3H), 5.21 (s, 3H), 4.58 (t, J = 4.7 Hz, 6H), 4.57 (s 6H), 3.95 (m, 9H), 3.85 to 3.92 (m, 9H), 3.74 to 3.80 (m, 9H), 3.71 (dd, J = 10.0) , 4.1 Hz, 3H), 3.55 to 3.68 (m, 42H), 3.25 (t, J = 6.5 Hz, 2H), 2.19 (t, J = 7.3 Hz, 2H) , 1.99 (s, 9H), 1.52-1.62 (m, 4H), 1.33-1.41 (m, 2H)

N- (1,3-bis [1- {1-[(1S, 2R, 3R, 4R, 5S) -4- (acetylamino) -2,3-dihydroxy-6,8-dioxabicyclo [3. 2.1] oct-1-yl] -2,5,8,11-tetraoxatridecan-13-yl} -1H-1,2,3-triazol-4-yl) methoxy] 2-{[( 1- {1-[(1S, 2R, 3R, 4R, 5S) -4- (acetylamino) -2,3-dihydroxy-6,8-dioxabicyclo [3.2.1] oct-1-yl ] -2,5,8,11-tetraoxatridecan-13-yl} -1H-1,2,3-triazol-4-yl) methoxy] methyl} propan-2-yl) hept-6-enamide ( 44)
In N, N-dimethylformamide (0.5 mL), N-[(1S, 2R, 3R, 4R, 5S) -1- (13- {4-[(3-[(1- {1-[( 1S, 2R, 3R, 4R, 5S) -4- (acetylamino) -2,3-dihydroxy-6,8-dioxabicyclo [3.2.1] oct-1-yl] -2,5,8 , 11-tetraoxatridecan-13-yl} -1H-1,2,3-triazol-4-yl) methoxy] -2-{[(1- {1-[(1S, 2R, 3R, 4R, 5S) -4- (acetylamino) -2,3-dihydroxy-6,8-dioxabicyclo [3.2.1] oct-1-yl] -2,5,8,11-tetraoxatridecane- 13-yl} -1H-1,2,3-triazol-4-yl} methoxy) methyl} -2-amino Ropoxy) methyl] -1H-1,2,3-triazol-1-yl} -2,5,8,11-tetraoxatridec-1-yl) -2,3-dihydroxy-6,8-dioxa To a solution of bicyclo [3.2.1] oct-4-yl] acetamide hydrochloride (42) (25 mg, 0.016 mmol) was added N, N-diisopropylethylamine (0.0111 mL, 0.0635 mmol). After stirring for 10 minutes, undiluted 1- (hept-6-enoyloxy) pyrrolidine-2,5-dione
(Angewandte Chemie, International Edition, 51 (25), 6144-6148, S6144 / 1-S6144 / 53, 2012, 5.36 mg, 0.0238 mmol) and the reaction was allowed to stir at room temperature for 18 hours. The reaction was then heated to 60 ° C. for 32 hours. After 32 hours, the reaction was concentrated under reduced pressure. The crude material was diluted with dimethyl sulfoxide (1 mL), passed through a syringe filter, and the crude material was purified by reverse phase chromatography using the following conditions to give the title compound as a gum (4.9 mg, 19%).

Purification conditions:
The residue was dissolved in dimethyl sulfoxide (1 mL) and purified by reverse phase HPLC. Column: Waters Sunfire C18, 19 × 100, 5u. Mobile phase A: 0.05% TFA / water (v / v). Mobile phase B: 0.05% TFA in acetonitrile (v / v). From 1% to 55% H ₂ O / 95% acetonitrile linearly to 55% H ₂ O / 45% acetonitrile, 0.5% to 55% H ₂ O / 45% acetonitrile linearly to 0% H ₂ O / 100% MeCN to 0% H ₂ O / 100% acetonitrile from 11.0 to 12.0 minutes. Flow rate: 25 mL / min.

QC condition:
Column: Waters Atlantis dC18, 4.6 × 50, 5u. Mobile phase A: 0.05% TFA in water (v / v). Mobile phase B: 0.05% TFA in acetonitrile (v / v). From 95.0% H ₂ O / 5.0% acetonitrile in 4.0 minutes to 5% H ₂ O / 95% acetonitrile in a straight line 5% H ₂ O / 95% acetonitrile from 4.0 minutes to 5.0 minutes Maintain. Flow rate: 2 mL / min. Retention time = 1.81. Observed mass = 825.2381. Method C: 3-minute operation LRMS [M-1 = 1647]. ¹ H-NMR (methanol-d ₄ ) δ: 7.9 (s, 3H), 5.69-5.88 (m, 1H), 5.21 (s, 3H), 4.95 (m, 2H) ), 4.51-4.63 (m, 12H), 3.95 (t, J = 9.7 Hz, 6H), 3.85-3.91 (m, 9H), 3.74-3.81 (M, 9H), 3.71 (dd, J = 9.4, 4.1 Hz, 3H), 3.54-3.68 (m, 42H), 2.17 (t, J = 7.3 Hz, 2H), 2.01-2.09 (m, 2H), 1.99 (s, 9H), 1.52-1.61 (m, 2H), 1.39 (quin, J = 7.5 Hz, 2H)

N- (1,3-bis [1- {1-[(1S, 2R, 3R, 4R, 5S) -4- (acetylamino) -2,3-dihydroxy-6,8-dioxabicyclo [3. 2.1] oct-1-yl] -2,5,8,11-tetraoxatridecan-13-yl} -1H-1,2,3-triazol-4-yl) methoxy] 2-{[( 1- {1-[(1S, 2R, 3R, 4R, 5S) -4- (acetylamino) -2,3-dihydroxy-6,8-dioxabicyclo [3.2.1] oct-1-yl ] -2,5,8,11-tetraoxatridecan-13-yl} -1H-1,2,3-triazol-4-yl) methoxy] methyl} propan-2-yl) hept-6-inamide ( 45)
In N, N-dimethylformamide (0.5 mL), N-[(1S, 2R, 3R, 4R, 5S) -1- (13- {4-[(3-[(1- {1-[( 1S, 2R, 3R, 4R, 5S) -4- (acetylamino) -2,3-dihydroxy-6,8-dioxabicyclo [3.2.1] oct-1-yl] -2,5,8 , 11-tetraoxatridecan-13-yl} -1H-1,2,3-triazol-4-yl) methoxy] -2-{[(1- {1-[(1S, 2R, 3R, 4R, 5S) -4- (acetylamino) -2,3-dihydroxy-6,8-dioxabicyclo [3.2.1] oct-1-yl] -2,5,8,11-tetraoxatridecane- 13-yl} -1H-1,2,3-triazol-4-yl} methoxy) methyl} -2-amino Ropoxy) methyl] -1H-1,2,3-triazol-1-yl} -2,5,8,11-tetraoxatridec-1-yl) -2,3-dihydroxy-6,8-dioxa To a solution of bicyclo [3.2.1] oct-4-yl] acetamide hydrochloride (42) (25 mg, 0.016 mmol) was added N, N-diisopropylethylamine (0.0111 mL, 0.0635 mmol). After stirring for 10 minutes, undiluted 1- (hept-6-inoyloxy) pyrrolidine-2,5-dione
(PCT International Application 2007056389, May 18, 2007: 5.31 mg, 0.0238 mmol) was added and the reaction was stirred at room temperature for 18 hours. The reaction was then heated to 60 ° C. for 32 hours. After 32 hours, the reaction was concentrated under reduced pressure. The crude material was diluted with dimethyl sulfoxide (1 mL), passed through a syringe filter, and the crude material was purified using reverse phase chromatography under the following conditions to give the title compound as a gum (5 mg, 19 %).

Purification conditions:
The residue was dissolved in dimethyl sulfoxide (1 mL) and purified by reverse phase HPLC (column: Waters Sunfire C18, 19 × 100, 5u). Mobile phase A: 0.05% TFA in water (v / v). Mobile phase B: 0.05% TFA in acetonitrile (v / v). 95% H ₂ O / 5.0% acetonitrile in 10.5 minutes linearly to 55% H ₂ O / 45% acetonitrile, 0.5% 55% H ₂ O / 45% acetonitrile in 0 minutes linearly Maintain 0% H ₂ O / 100% acetonitrile from 11.0 to 12.0 minutes to ₂ O / 100% MeCN. Flow rate: 25 mL / min.

QC condition:
Column: Waters Atlantis dC18, 4.6 × 50, 5u. Mobile phase A: 0.05% TFA in water (v / v). Mobile phase B: 0.05% TFA in acetonitrile (v / v). From 50.0% H ₂ O / 5.0% acetonitrile to 5% H ₂ O / 95% acetonitrile at 4.0 minutes, 5% H ₂ O / 95% acetonitrile maintained from 4.0 minutes to 5.0 minutes . Flow rate: 2 mL / min. Retention time = 1.68. Observed mass = 8244.2237. Method C: LRMS [1 / 2M = 823] running for 3 minutes. ¹ H-NMR (methanol-d ₄ ) δ: 7.9 (s, 3H), 5.21 (s, 3H), 4.59 (t, J = 5.0 Hz, 6H), 4.56 (s 6H), 3.95 (t, J = 10.0 Hz, 6H), 3.85-3.92 (m, 9H), 3.74-3.79 (m, 9H), 3.71 (dd , J = 10.0, 4.1 Hz, 3H), 3.54-3.67 (m, 41H), 2.13-2.24 (m, 6H), 1.99 (s, 9H), 1 .66 (quin, J = 7.5 Hz, 2H), 1.50 (quin, J = 7.3 Hz, 2H)

Ethyl 7-[(2,5-dioxopyrrolidin-1-yl) oxy] -7-oxoheptanoate (1-z-1)
7-Ethoxy-7-oxoheptanoic acid in N, N-dimethylformamide (6.0 mL)
To a solution of (448 mg, 2.38 mmol) was added N-hydroxysuccinimide (329 mg, 2.86 mmol) followed by N- (3-dimethylaminopropyl) -N′-ethylcarbodiimide hydrochloride (559 mg, 2.86 mmol). ) Was added. The reaction was stirred at room temperature for 72 hours. After 72 hours, the reaction was quenched with water and extracted three times with dichloromethane. The combined organic layers were washed with saturated sodium bicarbonate, brine, dried over sodium sulfate, filtered and concentrated under reduced pressure. The crude material was purified using CombiFlash Rf (RediSep 40 g gold column) eluting with a gradient of 0% to 100% ethyl acetate / heptane to give the title compound as a gum (426 mg 63%). Method C: LRMS [M + Na = 308] running for 1.5 minutes. ¹ H-NMR (methanol-d ₄ ) δ: 4.12 (q, J = 7.0 Hz, 2H), 2.83 (s, 4H), 2.64 (t, J = 7.2 Hz, 2H) 2.33 (t, J = 7.2 Hz, 2H), 1.74 (quin, J = 7.4 Hz, 2H), 1.58-1.68 (m, 2H), 1.40-1. 53 (m, 2H), 1.24 (t, J = 7.0 Hz, 3H).

7-[(1,3-bis [(1- {1-[(1S, 2R, 3R, 4R, 5S) -4- (acetylamino) -2,3-dihydroxy-6,8-dioxabicyclo [ 3.2.1] oct-1-yl] -2,5,8,11-tetraoxatridecan-13-yl} -1H-1,2,3-triazol-4-yl) methoxy] -2- {[(1- {1-[(1S, 2R, 3R, 4R, 5S) -4- (acetylamino) -2,3-dihydroxy-6,8-dioxabicyclo [3.2.1] octa- 1-yl] -2,5,8,11-tetraoxatridecan-13-yl} -1H-1,2,3-triazol-4-yl) methoxy] methyl} propan-2-yl) amino]- 7-Oxoheptanoic acid (sodium salt) (46)
In N, N-dimethylformamide (0.5 mL), N-[(1S, 2R, 3R, 4R, 5S) -1- (13- {4-[(3-[(1- {1-[( 1S, 2R, 3R, 4R, 5S) -4- (acetylamino) -2,3-dihydroxy-6,8-dioxabicyclo [3.2.1] oct-1-yl] -2,5,8 , 11-tetraoxatridecan-13-yl} -1H-1,2,3-triazol-4-yl) methoxy] -2-{[(1- {1-[(1S, 2R, 3R, 4R, 5S) -4- (acetylamino) -2,3-dihydroxy-6,8-dioxabicyclo [3.2.1] oct-1-yl] -2,5,8,11-tetraoxatridecane- 13-yl} -1H-1,2,3-triazol-4-yl} methoxy) methyl} -2-amino Ropoxy) methyl] -1H-1,2,3-triazol-1-yl} -2,5,8,11-tetraoxatridec-1-yl) -2,3-dihydroxy-6,8-dioxa To a solution of bicyclo [3.2.1] oct-4-yl] acetamide hydrochloride (42) (30.0 mg, 0.019 mmol) was added N, N-diisopropylethylamine (0.0133 mL, 00762 mmol), After stirring for 10 minutes, undiluted ethyl 7-[(2,5-dioxopyrrolidin-1-yl) oxy] -7-oxoheptanoate (Iz-1) (7.4 mg, 0.026 mmol) And the reaction was stirred at room temperature for 18 hours. The reaction was then heated to 60 ° C. for 32 hours. After 32 hours, the reaction was cooled to room temperature and concentrated under reduced pressure. The crude material was diluted with ethanol (1 mL) and water (0.03 mL), followed by the addition of 12.5 M aqueous sodium hydroxide (0.015 mL, 0.190 mmol). The reaction was stirred at room temperature for 3 hours. After 3 hours, the reaction was concentrated under reduced pressure. The crude material was diluted with dimethyl sulfoxide (1 mL) and filtered through a syringe filter. This solution was purified by reverse phase chromatography using the following conditions to give the title compound as a gum (3.7 mg, 11%).

Purification conditions The residue was dissolved in dimethyl sulfoxide (1 mL) and purified by reverse phase HPLC (column: Waters Sunfire C18, 19 × 100, 5u). Mobile phase A: 0.05% TFA in water (v / v)). Mobile phase B: 0.05% TFA in acetonitrile (v / v). 90.0% H ₂ O / 10.0% acetonitrile in 10.5 minutes linearly to 70% H ₂ O / 30% acetonitrile, 0.5 minutes in 70% H ₂ O / 30% acetonitrile linearly 0 Maintain 0% H ₂ O / 100% acetonitrile from 11.0 minutes to 12.0 minutes to% H ₂ O / 100% MeCN. Flow rate: 25 mL / min.

QC conditions Column: Waters Atlantis dC18, 4.6 × 50, 5u. Mobile phase A: 0.05% TFA in water (v / v). Mobile phase B: 0.05% TFA in acetonitrile (v / v). From 95.0% H ₂ O / 5.0% acetonitrile in 4.0 minutes to 5% H ₂ O / 95% acetonitrile in a straight line 5% H ₂ O / 95% acetonitrile from 4.0 minutes to 5.0 minutes Maintain. Flow rate: 2 mL / min. Retention time = 1.58 minutes. Observed mass = 839.7097). Method C: MassLynx \ Acid_3.0 Min. olp-LRMS [M + 1 = 1682]. ¹ H-NMR (methanol-d ₄ ) δ: 7.9 (s, 3H), 5.21 (s, 3H), 4.58 (t, J = 4.7 Hz, 6H), 4.56 (s 6H), 3.95 (t, J = 9.7 Hz, 6H), 3.89 (dt, J = 9.8, 4.8 Hz, 9H), 3.74-3.79 (m, 9H) 3.71 (dd, J = 10.0, 4.1 Hz, 3H), 3.53-3.67 (m, 42H), 2.25 (t, J = 7.3 Hz, 2H), 2. 17 (t, J = 7.3 Hz, 2H), 1.99 (s, 9H), 1.58 (dquin, J = 14.3, 7.3 Hz, 4H), 1.31-1.39 (m , 1H),

Benzyl {6- [1,3-bis [(1- {1-[(1S, 2R, 3R, 4R, 5S) -4- (acetylamino) -2,3-dihydroxy-6,8-dioxabicyclo [3.2.1] oct-1-yl] -2,5,8,11-tetraoxatridecan-13-yl} -1H-1,2,3-triazol-4-yl) methoxy] -2 -{[(1- {1-[(1S, 2R, 3R, 4R, 5S) -4- (acetylamino) -2,3-dihydroxy-6,8-dioxabicyclo [3.2.1] octa -1-yl] 2,5,8,11-tetraoxatridecan-13-yl} -1H-1,2,3-triazol-4-yl) methoxy] methyl} propan-2-yl) amino]- 6-oxohexyl} carbamate (47)
Benzyl {6-[(1,3-bis [(1- {1-[(1S, 2R, 6R, 7R, 8S) in acetic acid (6 mL), methanol (1.5 mL) and water (1.5 mL)]. ) -7- (acetylamino) -4,4-dimethyl-3,5,9,11-tetraoxatricyclo [6.2.1.0-2,6-] undec-1-yl] -2, 5,8,11-tetraoxatridecan-13-yl} -1H-1,2,3-triazol-4-yl) methoxy] -2-{[(1- {1-[(1S, 2R, 6R , 7R, 8S) -7- (acetylamino) -4,4-dimethyl-3,5,9,11-tetraoxatricyclo [6.2.1.0-2,6-] undec-1-yl ] -2,5,8,11-tetraoxatridecan-13-yl} -1H-1,2,3-triazole-4 Yl) methoxy] methyl} propan-2-yl) amino] -6-oxo-hexyl} carbamate (1-y-1) (308mg, a solution of 0.162 mmol), and heated for 64 hours to 70 ° C.. After 64 hours, the reaction was cooled to room temperature and concentrated under reduced pressure. The crude material was diluted with toluene and concentrated under reduced pressure. The crude material was diluted a second time with toluene and concentrated under reduced pressure to give the title compound (286 mg, none, 99%). Method C: LRMS running for 1.5 minutes [M + 1 = 1787]. ¹ H-NMR (methanol-d ₄ ) δ: 7.98 (s, 3H), 7.19-7.43 (m, 5H), 5.21 (s, 3H), 5.06 (s, 2H ), 4.50-4.66 (m, 12H), 3.95 (dd, J = 9.6, 5.7 Hz, 6H), 3.86-3.91 (m, 9H), 3.74. -3.78 (m, 9H), 3.71 (dd, J = 10.0, 4.1 Hz, 3H), 3.54-3.67 (m, 42H), 3.03-3.12 ( m, 2H), 2.11-2.24 (m, 2H), 1.98 (s, 9H), 1.51-1.63 (m, 2H), 1.43-1.51 (m, 2H), 1.33 (d, J = 6.6 Hz, 2H).

6-amino-N- (1,3-bis [(1- {1-[(1S, 2R, 3R, 4R, 5S) -4- (acetylamino) -2,3-dihydroxy-6,8-di Oxabicyclo [3.2.1] oct-1-yl] -2,5,8,11-tetraoxatridecan-13-yl} -1H-1,2,3-triazol-4-yl) methoxy] -2-{[(1- {1-[(1S, 2R, 3R, 4R, 5S) -4- (acetylamino) -2,3-dihydroxy-6,8-dioxabicyclo [3.2.1 ] Oct-1-yl] -2,5,8,11-tetraoxatridecan-13-yl} -1H-1,2,3-triazol-4-yl) methoxy] methyl} propan-2-yl) Hexanamide acetate (48)
Benzyl {6-[(1,3-bis [(1- {1-[(1S, 2R, 3R, 4R, 5S) -4- (acetylamino) -2,3-dihydroxy-6,8-dioxa Bicyclo [3.2.1] oct-1-yl] -2,5,8,11-tetraoxatridecan-13-yl} -1H-1,2,3-triazol-4-yl) methoxy]- 2-{[(1- {1-[(1S, 2R, 3R, 4R, 5S) -4- (acetylamino) -2,3-dihydroxy-6,8-dioxabicyclo [3.2.1] Octa-1-yl] 2,5,8,11-tetraoxatridecan-13-yl} -1H-1,2,3-triazol-4-yl) methoxy] methyl} propan-2-yl) amino] -6-oxohexyl} carbamate (47) (640 mg, 0.358 mm The l), was dissolved in methanol (20.0 mL) and acetic acid (0.041 mL, in 0.717 mmol). This solution was then used in the H cube with 10% palladium on carbon (small cartridge) using the following parameters (temperature = 50 ° C., flow rate = 1.0 mL / min, pressure = total H ₂ (1 bar)). Passed through. The solution was collected and concentrated under reduced pressure to give the title compound as a white foam (572 mg, 93%). Method C: LRMS [M + 1 = 1652] running for 3 minutes. ¹ H-NMR (methanol-d ₄ ) δ: 8.00 (s, 3H), 5.21 (s, 3H), 4.59 (t, J = 4.9 Hz, 6H), 4.56 (s 6H), 3.95 (d, J = 9.8 Hz, 6H), 3.85-3.92 (m, 9H), 3.74-3.79 (m, 9H), 3.69-3 .74 (m, 3H), 3.55-3.69 (m, 42H), 2.91 (t, J = 7.6 Hz, 2H), 2.20 (t, J = 7.2 Hz, 2H) , 1.99 (s, 9H), 1.90 (s, 3H), 1.52-1.68 (m, 4H), 1.34-1.43 (m, 2H)

N- {6-[(1,3-bis [(1- {1-[(1S, 2R, 3R, 4R, 5S) -4- (acetylamino) -2,3-dihydroxy-6,8-di Oxabicyclo [3.2.1] oct-1-yl] -2,5,8,11-tetraoxatridecan-13-yl} -1H-1,2,3-triazol-4-yl) methoxy] -2-{[(1- {1-[(1S, 2R, 3R, 4R, 5S) -4- (acetylamino) -2,3-dihydroxy-6,8-dioxabicyclo [3.2.1 ] Oct-1-yl] -2,5,8,11-tetraoxatridecan-13-yl} -1H-1,2,3-triazol-4-yl) methoxy] methyl} propan-2-yl) Amino] -6-oxohexyl} -6- (2,5-dioxo-2,5-dihydro-1H- Roll-1-yl) hexanamide (49)
6-amino-N- (1,3-bis [(1- {1-[(1S, 2R, 3R, 4R,) in N, N-dimethylformamide (0.5 mL) and tetrahydrofuran (0.5 mL). 5S) -4- (acetylamino) -2,3-dihydroxy-6,8-dioxabicyclo [3.2.1] oct-1-yl] -2,5,8,11-tetraoxatridecane- 13-yl} -1H-1,2,3-triazol-4-yl) methoxy] -2-{[(1- {1-[(1S, 2R, 3R, 4R, 5S) -4- (acetylamino) ) -2,3-dihydroxy-6,8-dioxabicyclo [3.2.1] oct-1-yl] -2,5,8,11-tetraoxatridecan-13-yl} -1H-1 , 2,3-Triazol-4-yl) methoxy] methyl} propane-2 Yl) hexanamide (48) (60 mg, 0.036 mmol) in a solution of N, N-diisopropylethylamine (0.0253 mL, 0.145 mmol) and 1- {6-[(2,5-dioxopyrrolidine-1 -Yl) oxy] -6-oxohexyl} -1H-pyrrole-2,5-dione
(12.3 mg, 0.040 mmol) was added at room temperature for 16 hours. After 16 hours, the reaction was concentrated under reduced pressure. The crude material was purified by reverse phase chromatography using the following conditions to give the title compound as a gum (15.4 mg, 23%).

Purification conditions The residue was dissolved in dimethyl sulfoxide (1 mL) and purified by reverse phase HPLC. Column: Waters Sunfire C18, 19 × 100, 5u. Mobile phase A: 0.05% TFA in water (v / v). Mobile phase B: 0.05% TFA in acetonitrile (v / v). Gradient: 80% H ₂ 0 / 20.0% acetonitrile in 10.5 minutes to 75% H ₂ O / 25% acetonitrile in a straight line to 0% H ₂ O / 100% MeCN up to 11.0 minutes, 11. Maintain 0% H ₂ O / 100% acetonitrile from 0 to 12.0 minutes. Flow rate: 25 mL / min.

QC conditions Column: Waters Atlantis dC18, 4.6 × 50, 5u. Mobile phase A: 0.05% TFA in water (v / v). Mobile phase B: 0.05% TFA in acetonitrile (v / v). Gradient: from 95% H ₂ O / 5.0% acetonitrile in 4.0 minutes to 5% H ₂ O / 95% acetonitrile with a _{linear, 5% H 2 O / 95} % acetonitrile to 4.0 min 5.0 min Maintain. Flow rate: 2 mL / min. Retention time = 1.69 minutes. Observed mass observation = 923.4907. Method C: 3-minute operation LRMS [M-1 = 1844]. ¹ H-NMR (methanol-d ₄ ) δ: 8.00 (s, 3H), 6.80 (s, 2H), 5.21 (s, 3H), 4.59 (t, J = 5.0 Hz) , 6H), 4.56 (s, 6H), 3.95 (t, J = 9.7 Hz, 6H), 3.90 (t, J = 5.0 Hz, 6H), 3.88 (d, J = 4.1 Hz, 3H), 3.74-3.79 (m, 9H), 3.71 (dd, J = 10.0, 4.1 Hz, 3H), 3.55-3.68 (m, 42H), 3.48 (t, J = 7.0 Hz, 2H), 3.12 (t, J = 7.0 Hz, 2H), 2.11-2.23 (m, 4H), 1.99 ( s, 9H), 1.53-1.66 (m, 6H), 1.48 (quin, J = 7.2 Hz, 2H), 1.24-1.36 (m, 4H)

N- {6- [1,3-bis [(1- {1-[(1S, 2R, 3R, 4R, 5S) -4- (acetylamino) -2,3-dihydroxy-6,8-dioxabicilo [ 3.2.1] oct-1-yl] -2,5,8,11-tetraoxatridecan-13-yl} -1H-1,2,3-triazol-4-yl) methoxy] -2- {[(1- {1-[(1S, 2R, 3R, 4R, 5S) -4- (acetylamino) -2,3-dihydroxy-6,8-dioxabicyclo [3.2.1] octa- 1-yl] -2,5,8,11-tetraoxatridecan-13-yl} -1H-1,2,3-triazol-4-yl) methoxy] methyl} propan-2-yl) amino]- 6-oxohexyl} -6-[(bromoacetyl) amino] hexanamide (50
6-amino-N- (1,3-bis [(1- {1-[(1S, 2R, 3R, 4R,) in N, N-dimethylformamide (0.5 mL) and tetrahydrofuran (0.5 mL). 5S) -4- (acetylamino) -2,3-dihydroxy-6,8-dioxabicyclo [3.2.1] oct-1-yl] -2,5,8,11-tetraoxatridecane- 13-yl} -1H-1,2,3-triazol-4-yl) methoxy] -2-{[(1- {1-[(1S, 2R, 3R, 4R, 5S) -4- (acetylamino) ) -2,3-dihydroxy-6,8-dioxabicyclo [3.2.1] oct-1-yl] -2,5,8,11-tetraoxatridecan-13-yl} -1H-1 , 2,3-Triazol-4-yl) methoxy] methyl} propane-2 Yl) hexanamide (48) (60 mg, to a solution of 0.036 mmol), N, N-diisopropylethylamine (0.0253mL, 0.145mmol) and 6 - [(bromoacetyl) amino] hexanoic acid pentafluorophenyl
(Chemistr-A European Journal, 14 (16), 4939-4947, 2008, 16.7 mg, 0.0400 mmol) was added at room temperature for 16 hours. After 16 hours, the reaction was concentrated under reduced pressure. The crude material was purified using reverse phase chromatography under the following conditions to give the title compound as a gum (4.4 mg, 6.4%). Observed mass: 944.1543.

Purification conditions The residue was dissolved in dimethyl sulfoxide (1 mL) and purified by reverse phase HPLC. Column: Waters Sunfire C18, 19 × 100, 5u. Mobile phase A: 0.05% TFA in water (v / v). Mobile phase B: 0.05% TFA in acetonitrile (v / v). Gradient: 80% H ₂ O / 20.0% acetonitrile in 10.5 minutes to 75% H ₂ O / 25% acetonitrile in a straight line to 0% H ₂ O / 100% MeCN up to 11.0 minutes, 11. Maintain 0% H ₂ O / 100% acetonitrile from 0 to 12.0 minutes. Flow rate: 25 mL / min.

QC conditions Column: Waters Atlantis dC18, 4.6 × 50, 5u. Mobile phase A: 0.05% TFA in water (v / v). Mobile phase B: 0.05% TFA in acetonitrile (v / v). Gradient: from 95% H ₂ O / 5.0% acetonitrile in 4.0 minutes to 5% H ₂ O / 95% acetonitrile with a _{linear, 5% H 2 O / 95} % acetonitrile to 4.0 min 5.0 min Maintain. Flow rate: 2 mL / min. Retention time = 1.64 minutes. Observed mass = 944.1543. Method C: 3-minute run LRMS [M + 1 = 1886]. ¹ H-NMR (methanol-d ₄ ) δ: 7.9 (s, 3H), 5.21 (s, 3H), 4.57-4.62 (m, 6H), 4.56 (s, 6H) ), 3.92-3.98 (m, 6H), 3.83-3.91 (m, 10H), 3.80 (s, 2H), 3.69-3.79 (m, 12H), 3.54-3.68 (m, 43H), 3.13 (t, J = 6.7 Hz, 2H), 2.18 (d, J = 6.5 Hz, 4H), 1.98 (s, 9H) ), 1.59-1.67 (m, 2H), 1.51-1.59 (m, 4H), 1.48 (br.s., 2H), 1.27-1.41 (m, 4H)

9H-Fluoren-9-ylmethyl {(1S) -1-cyclopentyl-2-[(2,5-dioxopyrrolidin-1-yl) oxy] -2-oxoethyl} carbamate (1-aa-1)
N, N′-dicyclohexylcarbodiimide (247 mg, 1.2 mmol) was added to (2S) -cyclopentyl {[(9H-fluoren-9-ylmethoxy) carbonyl] amino} ethane in dehydrated tetrahydrofuran (40 mL) at 5-10 ° C. acid
(380 mg, 1.04 mmol) and N-hydroxysuccinimide (137.6 mg, 1.2 mmol) were added in portions. After the addition, the mixture was stirred overnight at room temperature. The mixture was cooled to -20 ° C and then filtered to remove by-products. The filter cake was washed with cold tetrahydrofuran and the filtrate was concentrated to dryness and purified on a flash column (eluting with petroleum ether: ethyl acetate 100: 10 to 100: 50) to give the title compound (380 mg, 79%). .

N-5-carbamoyl-N-2-[(2S) -2-cyclopentyl-2-{[(9H-fluoren-9-ylmethoxy) carbonyl] amino} acetyl] -L-ornithine (1-ab-1 )
To a solution of (2S) -2-amino-5- (carbamoylamino) pentanoic acid (151 mg, 0.86 mmol) and sodium bicarbonate (72.5 mg, 0.86 mmol) in water (15 mL) was added tetrahydrofuran (10 mL). Was added at 0 ° C. The resulting mixture was added 9H-fluoren-9-ylmethyl {(1S) -1-cyclopentyl-2-[(2,5-dioxopyrrolidin-1-yl) in 1,2-dimethoxy-ethane (15 mL). A solution of oxy] -2-oxoethyl} carbamate (1-aa-1) (380 mg, 0.82 mmol) was added dropwise under nitrogen. After the addition, the mixture was stirred overnight at room temperature. The reaction mixture was washed 4 times with methyl-tert-butyl ether (50 mL). The organic phase was discarded and the aqueous layer was acidified to pH = 3-4 with aqueous hydrochloric acid (1M). This solution was extracted 6 times with chloroform / isopropyl alcohol (4: 1) (50 mL). The combined organic layers were dried over sodium sulfate and concentrated to dryness to give the title compound as a white solid (403 mg, 93.7%).

9H-Fluoren-9-ylmethyl [(1S) -2-{[(2S) -5- (carbamoylamino) -1-{[4- (hydroxymethyl) phenyl] amino} -1-oxopentan-2-yl Amino} -1-cyclopentyl-2-oxoethyl] carbamate (1-ac-1)
N-5-carbamoyl-N-2-2-[(2S) -2-cyclopentyl-2-{[(9H-fluoren-9-ylmethoxy) carbonyl] amino} acetyl in dichloromethane / methanol (30 mL / 15 mL) ] To a solution of -L-ornithine (1-ab-1) (500 mg, 0.95 mmol) and 4-aminobenzyl alcohol (470 mg, 3.82 mmol) was added N-ethoxycarbonyl-2-ethoxy-1,2-dihydro. Quinoline (708 mg, 2.86 mmol) was added. The reaction mixture was then stirred overnight at room temperature in the dark. The next morning, the reaction was concentrated under reduced pressure and the residue was washed with methyl tert-butyl ether (100 mL × 3). The filter cake was then purified by preparative HPLC (see conditions below) to give the title compound as a yellow solid (31 mg, 5.1%). ¹ H-NMR (400 MHz, DMSO) δ: 9.95 (s, 1H), 8.09 (d, 1H), 7.90-7.88 (d, 2H), 7.73-7.71 ( t, 2H), 7.55-7.53 (t, 2H), 7.41 (t, 2H), 7.34-7.30 (t, 2H), 7.24-7.22 (d, 2H), 5.96 (t, 1H), 5.39 (s, 2H), 5.11-5.08 (t, 1H), 4.44-4.42 (d, 3H), 4.32. -4.23 (m, 3H), 3.96-3.92 (t, 1H), 3.01-3.00 (m, 3H), 2.15-2.13 (m, 1H), 1 .66-1.24 (m, 12H), m / z for C ₃₅ H ₄₁ N ₅ O ₆ : 628.4 (M + H) ⁺ , retention time: 4.213 minutes

Purification conditions Column: DIKMA Diasilsil (2) C18, 200 * 20 mm * 5 um. Mobile phase: 30% acetonitrile in water (0.1% TFA) to 50% acetonitrile in water (0.1% TFA). Wavelength = 220 nm. Work-up: Concentrate and freeze-dry.

QC condition:
Column: Ultimate XB-C18, 3 * 50 mm, 3 um. Retention time: 4.33 minutes. Mobile phase: A, water (2.7 mL TFA in 4 L water). B: Acetonitrile (2.5 mL TFA in 4 L acetonitrile), elution gradient 1% to 100%. Wavelength: 220 nm. ee value: 100%. Column: Chiralcel OD-3, 50 * 4.6 mm, I.D. D. 3um. Retention time: 1.923 minutes. Mobile phase: 5% to 40% ethanol in CO ₂ (0.05% DEA). Flow rate: 2.5 mL / min. Wavelength: 254 nm. ee value = 100%. Column: AD-3, 50 * 4.6 mm, I.D. D. 3 μm. Retention time: 1.981 minutes. Mobile phase: 5% to 40% ethanol in CO ₂ (0.05% DEA). Flow rate: 2.5 mL / min. Wavelength: 220nm

N ~ 2-[(2S) -2-amino-2-cyclopentylacetyl] -N ~ 5-carbamoyl-N- [4- (hydroxymethyl) phenyl] -L-ornithine amide (1-ad-1)
9H-Fluoren-9-ylmethyl [(1S) -2-{[(2S) -5- (carbamoylamino) -1-{[4- (hydroxymethyl) phenyl] in N, N-dimethylformamide (10 mL). ] Amino} -1-oxopentan-2-yl] amino} -1-cyclopentyl-2-oxoethyl] carbamate (1-ac-1) (500 mg, 0.797 mmol) in a stirred solution at 5 ° C. under nitrogen. Piperidine (4 mL) was added dropwise. The mixture was stirred at room temperature for 1.5 hours. The reaction was concentrated to dryness. The crude product was washed with dichloromethane (20 mL), filtered, and the filter cake was dried in vacuo to give the title compound (300 mg, 93.1%) as a solid that was taken to the next step without purification. used.

N-[(1S) -2-{[(2S) -5- (carbamoylamino) -1-{[4- (hydroxymethyl) phenyl] amino} -1-oxopentan-2-yl] amino} -1 -Cyclopentyl-2-oxoethyl] -6- (2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl) hexanamide (1-ae-1)
N ~ 2-[(2S) -2-amino-2-cyclopentylacetyl] -N-5-carbamoyl-N- [4- (hydroxymethyl) phenyl] -L in N, N-dimethylformamide (12 mL) -To a stirred solution of ornithine amide (1-ad-1) (300 mg, 0.74 mmol), 1- {6-[(2,5-dioxopyrrolidin-1-yl) oxy] -6-oxohexyl}- 1H-pyrrole-2,5-dione (272 mg, 0.889 mmol) was added at 3 ° C. under nitrogen. The mixture was stirred at room temperature for 2 hours. The reaction was added dropwise to methyl tert-butyl ether (250 mL), stirred at room temperature for 20 minutes, filtered, and the filter cake was concentrated to dryness to give the title compound (300 mg, 67.8%) as a solid. This was used in the next step without purification.

N-5--carbamoyl-N-2-[(2S) -2-cyclopentyl-2-{[6- (2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl) hexanoyl] Amino} acetyl] -N- [4-({[(4-nitrophenoxy) carbonyl] oxy} methyl) phenyl] -L-ornithine amide (1-af-1)
N-[(1S) -2-{[(2S) -5- (carbamoylamino) -1-{[4- (hydroxymethyl) phenyl] amino} -1 in N, N-dimethylformamide (12 mL) -Oxopentan-2-yl] amino} -1-cyclopentyl-2-oxoethyl] -6- (2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl) hexanamide (1-ae- 1) To a stirred solution of (300 mg, 0.740 mmol) was added bis (4-nitrophenyl) carbonate (900 mg, 2.96 mmol) and N, N-diisopropylethylamine (390 mg, 2.96 mmol) at 3 ° C. under nitrogen. Added. The reaction was stirred overnight at room temperature. The reaction was added dropwise to methyl tert-butyl ether (60 mL), stirred at room temperature for 20 minutes, filtered, and the filter cake was washed with methyl tert-butyl ether (100 mL). This crude product was dried in vacuo to dryness. The crude product was purified by flash column eluting with 100: 1 to 94: 6 dichloromethane: methanol to give the title compound as a solid (50 mg, 17.7%). ¹ H-NMR (400 MHz, DMSO) δ: 10.09 (br, 1H), 8.33 (d, 2H), 8.13 (d, 1H), 7.93 (d, 1H), 7.67 -7.41 (m, 6H), 7.01 (s, 2H), 5.98 (br, 1H), 5.43 (s, 2H), 5.25 (s, 2H), 4.39 ( m, 1H), 4.23-4.19 (m, 1H), 3.37 (m, 1H), 3.03-2.96 (m, 2H), 2.14-2.11 (m, 3H), 1.70-1.19 (m, 19H). _{LC-MS: C 37 H 45} N 7 for O ₁₁ of m / z: 764.3 (M + H) +. Retention time: 0.823 minutes.

4-{[(2R) -5- (carbamoylamino) -2-{[(2R) -2-cyclopentyl-2-{[6- (2,5-dioxo-2,5-dihydro-1H-pyrrole- 1-yl) hexanoyl] amino} acetyl] amino} pentanoyl] amino} benzyl {6-[(1,3-bis [(1- {1-[(1S, 2R, 3R, 4R, 5S) -4- ( Acetylamino) -2,3-dihydroxy-6,8-dioxabicyclo [3.2.1] oct-1-yl] -2,5,8,11-tetraoxatridecan-13-yl} -1H -1,2,3-triazol-4-yl) methoxy] -2-{[(1- {1-[(1S, 2R, 3R, 4R, 5S) -4- (acetylamino) -2,3- Dihydroxy-6,8-dioxabicyclo [3.2.1] octa-1 Yl] -2,5,8,11-tetraoxatridecan-13-yl} -1H-1,2,3-triazol-4-yl) methoxy] methyl} propan-2-yl) amino] -6 Oxohexyl} carbamate (51)
6-Amino-N- (1,3-bis [(1- {1-[(1S, 2R, 3R, 4R,) in N, N-dimethylformamide (0.5 mL) and tetrahydrofuran (0.3 mL). 5S) -4- (acetylamino) -2,3-dihydroxy-6,8-dioxabicyclo [3.2.1] oct-1-yl] -2,5,8,11-tetraoxatridecane- 13-yl} -1H-1,2,3-triazol-4-yl) methoxy] -2-{[(1- {1-[(1S, 2R, 3R, 4R, 5S) -4- (acetylamino) ) -2,3-dihydroxy-6,8-dioxabicyclo [3.2.1] oct-1-yl] -2,5,8,11-tetraoxatridecan-13-yl} -1H-1 , 2,3-Triazol-4-yl) methoxy] methyl} propane-2 Yl) hexanamide (48) (45 mg, 0.027 mmol) in a solution of N, N-diisopropylethylamine (0.019 mL, 0.109 mmol) and N-5-carbamoyl-N-2 for 18 hours at room temperature. -[(2S) -2-cyclopentyl-2-{[6- (2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl) hexanoyl] amino} acetyl] -N- [4- ( {[(4-Nitrophenoxy) carbonyl] oxy} methyl) phenyl] -L-ornithinamide (1-af-1) (20.8 mg, 0.0272 mmol) was added. The sample was removed after 18 hours and UPLC showed the formation of the desired product. The crude reaction mixture was concentrated under reduced pressure. The resulting crude material was purified by reverse phase chromatography using the following conditions to give the title compound as a gum (21.7 mg, 35%).

Purification conditions:
The residue was dissolved in dimethyl sulfoxide (1 mL) and purified by reverse phase HPLC. Column: Waters Sunfire C18, 19 × 100, 5u. Mobile phase A: 0.05% TFA in water (v / v). Mobile phase B: 0.05% TFA in acetonitrile (v / v). Gradient: from 75.0% H ₂ O / 25.0% acetonitrile in 10.5 minutes to 65% H ₂ O / 35% acetonitrile in a straight line to 0% H ₂ O / 100% MeCN up to 11.0 minutes, Maintain 0% H ₂ O / 100% acetonitrile from 11.0 to 12.0 minutes. Flow rate: 25 mL / min.

QC condition:
Column: Waters Atlantis dC18, 4.6 × 50, 5u. Mobile phase A: 0.05% TFA in water (v / v). Mobile phase B: 0.05% TFA in acetonitrile (v / v). From 95.0% H ₂ O / 5.0% acetonitrile in 4.0 minutes to 5% H ₂ O / 95% acetonitrile in a straight line 5% H ₂ O / 95% acetonitrile from 4.0 minutes to 5.0 minutes Maintain. Flow rate: 2 mL / min. Retention time = 1.99 minutes. Observed mass = 11399.1254. Method C: LRMS [1 / 2M = 1138] running for 1.5 minutes. ¹ H-NMR (methanol-d ₄ ) δ: 7.9 (s, 3H), 7.57 (d, J = 8.2 Hz, 2H), 7.30 (d, J = 8.2 Hz, 2H) , 6.79 (s, 2H), 5.21 (s, 3H), 5.01 (s, 2H), 4.55-4.64 (m, 12H), 4.51 (dd, J = 9 0.0, 5.1 Hz, 1H), 4.43 (q, J = 7.2 Hz, 1H), 4.16 (d, J = 9.4 Hz, 1H), 3.95 (d, J = 9. 8 Hz, 6H), 3.85-3.91 (m, 9H), 3.74-3.79 (m, 9H), 3.71 (dd, J = 9.8, 4.3 Hz, 3H), 3.54-3.67 (m, 41H), 3.47 (t, J = 7.0 Hz, 2H), 3.16-3.26 (m, 1H), 3.10-3.16 (m , 1 ), 3.07 (t, J = 6.8 Hz, 2H), 2.24 (q, J = 7.7 Hz, 3H), 2.16 (t, J = 7.4 Hz, 2H), 1.99 (S, 9H), 1.85-1.95 (m, 1H), 1.42-1.84 (m, 16H), 1.37 (t, J = 7.0 Hz, 2H), 1.23 -1.34 (m, 5H)

N- (1,3-bis [(1- {1-[(1S, 2R, 3R, 4R, 5S) -4- (acetylamino) -2,3-dihydroxy-6,8-dioxabicyclo [3 2.1] oct-1-yl] -2,5,8,11-tetraoxatridecan-13-yl} -1H-1,2,3-triazol-4-yl) methoxy] -2- { [(1- {1-[(1S, 2R, 3R, 4R, 5S) -4- (acetylamino) -2,3-dihydroxy-6,8-dioxabicyclo [3.2.1] octa-1 -Yl] -2,5,8,11-tetraoxatridecan-13-yl} -1H-1,2,3-triazol-4-yl) methoxy] methyl} propan-2-yl) -3,19 -Dioxo-1- (pyridin-2-yldisulfanyl) -7,10,13,16-te Raokisa 4,20 diazacarbazole hexa concentrated acid -26- amide (52)
6-Amino-N- (1,3-bis [(1- {1-[(1S, 2R, 3R, 4R,) in N, N-dimethylformamide (0.6 mL) and tetrahydrofuran (0.6 mL). 5S) -4- (acetylamino) -2,3-dihydroxy-6,8-dioxabicyclo [3.2.1] oct-1-yl] -2,5,8,11-tetraoxatridecane- 13-yl} -1H-1,2,3-triazol-4-yl) methoxy] -2-{[(1- {1-[(1S, 2R, 3R, 4R, 5S) -4- (acetylamino) ) -2,3-dihydroxy-6,8-dioxabicyclo [3.2.1] oct-1-yl] -2,5,8,11-tetraoxatridecan-13-yl} -1H-1 , 2,3-Triazol-4-yl) methoxy] methyl} propane-2 Yl) hexanamide acetate (48) (70.0 mg, 0.041 mmol) and N- {15-[(2,5-dioxopyrrolidin-1-yl) oxy] -15-oxo-3,6,9 , 12-Tetraoxapentadec-1-yl} -3- (pyridin-2-yl) propanamide
To a solution of (27.5 mg, 0.0491 mmol), N, N-diisopropylethylamine (0.0285 mL, 0.164 mmol) was added. The reaction was stirred at room temperature for 18 hours. After 18 hours, the reaction was concentrated under reduced pressure. The crude material was purified by reverse phase chromatography using the following conditions to give the title compound as a gum (47.7 mg, 56%). Method C: 3-minute run LRMS [1 / 3M + 1 = 699]. ¹ H-NMR (methanol-d ₄ ) δ: 8.47 (d, J = 4.7 Hz, 1H), 8.01 (s, 3H), 7.93 (d, J = 3.5 Hz, 2H) 7.30-7.38 (s, 1H), 5.21 (s, 3H), 4.57-4.62 (m, 6H), 4.57 (s, 6H), 3.92-3 .99 (m, 6H), 3.89 (dd, J = 10.7, 4.9 Hz, 9H), 3.74-3.80 (m, 9H), 3, 72 (d, J = 9. 8, 4.7 Hz, 6H), 3.51-3.68 (m, 55H), 3.35-3.41 (m, 2H), 3.14 (t, J = 7.0 Hz, 2H), 3.10 (t, J = 6.8 Hz, 2H), 2.64 (t, J = 7.0 Hz, 2H), 2.43 (t, J = 6.0 Hz, 2H), 2.17 (t , J = .4 Hz, 2H), 1.99 (s, 9H), 1.52-1.61 (m, 2H), 1.43-1.51 (m, 2H), 1.27-1.38 (m) , 2H)

Purification conditions The residue was dissolved in dimethyl sulfoxide (1 mL) and purified by reverse phase HPLC. Column: Waters Sunfire C18, 19 × 100, 5u. Mobile phase A: 0.05% TFA in water (v / v). Mobile phase B: 0.05% TFA in acetonitrile (v / v). Gradient: 80.0% H ₂ O / 20.0% acetonitrile in 8.5 minutes to 70% H ₂ O / 30% acetonitrile in a straight line, 0% H ₂ O / 100% MeCN up to 9.0 minutes, Maintain 0% H ₂ O / 100% acetonitrile from 9.0 to 10.0 minutes. Flow rate: 25 mL / min.

QC conditions Column: Waters Atlantis dC18, 4.6 × 50, 5u. Mobile phase A: 0.05% TFA in water (v / v). Mobile phase B: 0.05% TFA in acetonitrile (v / v). Gradient: 95.0% H ₂ O / 5.0% acetonitrile at 4.0 minutes to 5% H ₂ O / 95% acetonitrile linearly from 4.0 minutes to 5.0% 5% H ₂ O / 95 % Acetonitrile maintained. Flow rate: 2 mL / min. Retention time = 1.78 minutes. Observed mass = 699.6404

N- (1,3-bis [(1- {1-[(1S, 2R, 3R, 4R, 5S) -4- (acetylamino) -2,3-dihydroxy-6,8-dioxabicyclo [3 2.1] oct-1-yl] -2,5,8,11-tetraoxatridecan-13-yl} -1H-1,2,3-triazol-4-yl) methoxy] -2- { [(1- {1-[(1S, 2R, 3R, 4R, 5S) -4- (acetylamino) -2,3-dihydroxy-6,8-dioxabicyclo [3.2.1] octa-1 -Yl] -2,5,8,11-tetraoxatridecan-13-yl} -1H-1,2,3-triazol-4-yl) methoxy] methyl} propan-2-yl) -3,31 -Dioxo-1- (pyridin-2-yldisulfanyl) -7,10,13,16,1 , 22,25,28- Okutaokisa -4,32- diazacarbazole octa Tria near end -38- amide (53)
6-Amino-N- (1,3-bis [(1- {1-[(1S, 2R, 3R, 4R,) in N, N-dimethylformamide (0.6 mL) and tetrahydrofuran (0.6 mL). 5S) -4- (acetylamino) -2,3-dihydroxy-6,8-dioxabicyclo [3.2.1] oct-1-yl] -2,5,8,11-tetraoxatridecane- 13-yl} -1H-1,2,3-triazol-4-yl) methoxy] -2-{[(1- {1-[(1S, 2R, 3R, 4R, 5S) -4- (acetylamino) ) -2,3-dihydroxy-6,8-dioxabicyclo [3.2.1] oct-1-yl] -2,5,8,11-tetraoxatridecan-13-yl} -1H-1 , 2,3-Triazol-4-yl) methoxy] methyl} propane-2 Yl) hexanamide acetate (48) (70.0 mg, 0.041 mmol) and N- {27-[(2,5-dioxopyrrolidin-1-yl) oxy] -27-oxo-3,6,9 , 12,15,18,21,24-octaoxaheptacos-1-yl} -3- (pyridin-2-yldisulfanyl) propanamide
To a solution of (30.1 mg, 0.041 mmol) was added N, N-diisopropylethylamine (0.0285 mL, 0.164 mmol). The reaction was stirred at room temperature for 18 hours. After 18 hours, the reaction was concentrated under reduced pressure. The crude material was purified by reverse phase chromatography using the following conditions to give the title compound as a gum (59.2 mg, 64%). Method C: 3-minute operation LRMS [1 / 3M = 757]. ¹ H-NMR (methanol-d ₄ ) δ: 8.47 (d, J = 5.1 Hz, 1H), 8.01 (s, 3H), 7.92 (d, J = 3.5 Hz, 2H) , 7.30-7.39 (m, 6H), 5.21 (s, 3H), 4.57 (s, 6H), 3.92-3.99 (m, 6H), 3.86-3 .92 (m, H), 3.74 (m, 6H), 3.77 (m, 9H), 3.69-3.74 (m, 6H), 3.50-3.68 (m, 73H) ), 3.14 (t, J = 7.0 Hz, 2H), 3.10 (t, J = 7.0 Hz, 2H), 2.64 (t, J = 6.8 Hz, 2H), 2.43 (T, J = 6.0 Hz, 2H), 2.17 (t, J = 7.4 Hz, 2H), 1.99 (s, 9H), 1.53-1.63 (m, 2H), 1 .42-1. 52 (m, 2H), 1.32 (dt, J = 15.1, 7.5 Hz, 2H)

Purification conditions The residue was dissolved in dimethyl sulfoxide (1 mL) and purified by reverse phase HPLC. Column: Waters Sunfire C18, 19 × 100, 5u. Mobile phase A: 0.05% TFA in water (v / v). Mobile phase B: 0.05% TFA in acetonitrile (v / v). Gradient: 80.0% H ₂ O / 20.0% acetonitrile in 8.5 minutes to 70% H ₂ O / 30% acetonitrile in a straight line, 0% H ₂ O / 100% MeCN up to 9.0 minutes, Maintain 0% H ₂ O / 100% acetonitrile from 9.0 to 10.0 minutes. Flow rate: 25 mL / min.

QC conditions Column: Waters Atlantis dC18, 4.6 × 50, 5u. Mobile phase A: 0.05% TFA in water (v / v). Mobile phase B: 0.05% TFA in acetonitrile (v / v). Gradient: from 95% H ₂ O / 5.0% acetonitrile in 4.0 minutes to 5% H ₂ O / 95% acetonitrile with a _{linear, 5% H 2 O / 95} % acetonitrile to 4.0 min 5.0 min Maintain. Flow rate: 2 mL / min. Retention time = 1.85 minutes. Observed mass = 758.405

6-amino-N- (1,3-bis [(1- {1-[(1S, 2R, 6R, 7R, 8S) -7- (acetylamino) -4,4-dimethyl-3,5,9 , 11-tetraoxatricyclo [6.2.1.0-2,6-] undec-1-yl] -2,5,8,11-tetraoxatridecan-13-yl} -1H-1, 2,3-triazol-4-yl) methoxy] -2-{[(1- {1-[(1S, 2R, 6R, 7R, 8S) -7- (acetylamino) -4,4-dimethyl-3 , 5,9,11-tetraoxatricyclo [6.2.1.0-2,6-] undec-1-yl] -2,5,8,11-tetraoxatridecan-13-yl}- 1H-1,2,3-triazol-4-yl) methoxy] methyl} propan-2-yl) hexanamide (1- g-1)
Benzyl- {6-[(1,3-bis [(1- {1-[(1S, 2R, 6R, 7R, 8S) -7- (acetylamino) -4,4-dimethyl-3,5,9 , 11-tetraoxatricyclo [6.2.1.0-2,6-] undec-1-yl] -2,5,8,11-tetraoxatridecan-13-yl} -1H-1, 2,3-triazol-4-yl) methoxy] -2-{[(1- {1-[(1S, 2R, 6R, 7R, 8S) -7- (acetylamino) -4,4-dimethyl-3 , 5,9,11-tetraoxatricyclo [6.2.1.0-2,6-] undec-1-yl] -2,5,8,11-tetraoxatridecan-13-yl}- 1H-1,2,3-triazol-4-yl) methoxy] methyl} propan-2-yl) amino] -6-oxy Hexyl} carbamate (1-y-1) to (1200mg, 0.63mmol), was dissolved in methanol (30 mL). This solution was then used with 10% palladium on carbon (small cartridge) using the following parameters (temperature = 50 ° C., flow rate = 1.0 mL / min, pressure = total H ₂ (1 bar)). Passed through H-cube. This solution was collected. When a sample was removed, UPLC showed that starting material remained. The reaction was passed through the H-cube a second time using the parameters described above. The collected solution was concentrated under reduced pressure to give the title compound as a white foam (1039 mg, 93%). Method C: LRMS [1 / 2M = 886] running for 1.5 minutes. ¹ H-NMR (methanol-d ₄ ) δ: 7.9 (s, 3H), 5.23 (d, J = 1.6 Hz, 3H), 4.45-4.62 (m, 12H), 4 .29 (d, J = 5.9 Hz, 3H), 4.16 (t, J = 6.4 Hz, 3H), 3.87-3.98 (m, 12H), 3.73-3.85 ( m, 15H), 3.54-3.70 (m, 36H), 2.87 (t, J = 7.6 Hz, 2H), 2.20 (t, J = 7.2 Hz, 2H), 98 (s, 9H), 1.53-1.69 (m, 4H), 1.48 (s, 9H), 1.34-1.41 (m, 2H), 1.33 (s, 9H)

N- {6-[(1,3-bis [(1- {1-[(1S, 2R, 6R, 7R, 8S) -7- (acetylamino) -4,4-dimethyl-3,5,9 , 11-tetraoxatricyclo [6.2.1.0-2,6-] undec-1-yl] -2,5,8,11-tetraoxatridecan-13-yl} -1H-1, 2,3-triazol-4-yl) methoxy] -2-{[(1- {1-[(1S, 2R, 6R, 7R, 8S) -7- (acetylamino) -4,4-dimethyl-3 , 5,9,11-tetraoxatricyclo [6.2.1.0-2,6-] undec-1-yl] -2,5,8,11-tetraoxatridecan-13-yl}- 1,2,3-triazol-4-yl) methoxy] methyl} propan-2-yl) amino] -6-oxohexyl} 6- (pyridin-2-yldisulfanyl) hexanamide (1-ag-2)
6-Amino-N- (1,3-bis [(1- {1-[(1S, 2R, 6R, 7R,) in N, N-dimethylformamide (0.5 mL) and tetrahydrofuran (0.5 mL). 8S) -7- (acetylamino) -4,4-dimethyl-3,5,9,11-tetraoxatricyclo [6.2.1.0-2,6-] undec-1-yl] -2 , 5,8,11-tetraoxatridecan-13-yl} -1H-1,2,3-triazol-4-yl) methoxy] -2-{[(1- {1-[(1S, 2R, 6R, 7R, 8S) -7- (Acetylamino) -4,4-dimethyl-3,5,9,11-tetraoxatricyclo [6.2.1.0-2,6-] undec-1- Yl] -2,5,8,11-tetraoxatridecan-13-yl} -1H-1,2,3 To a solution of triazol-4-yl) methoxy] methyl} propan-2-yl) hexanamide (1-ag-1) (105.0 mg, 0.0593 mmol) was added N, N-diisopropylethylamine (0.031 mL, 0 .178 mmol) and stirred for 10 minutes, then 1-{[6- (pyridin-2-yldisulfanyl) hexanoyl] oxy} pyrrolidine-2,5-dione (1-s-1) (25.2 mg, 0.0711 mmol) was added and the reaction was heated to room temperature for 16 hours. After 16 hours, the reaction was diluted with water (15 mL) and brine (5 mL) and extracted three times with dichloromethane (20 mL). The combined organic layers were washed with water (20 mL), brine (20 mL), dried over magnesium sulfate, filtered and concentrated under reduced pressure. The crude material was purified using CombiFlash Rf (RediSep 12 g silica gel column) eluting with a methanol / dichloromethane gradient from 0 to 20% to give the title compound (56.6 mg, 50%). Method C: MassLynx \ Acid_3.0 Min. olp-LRMS [1 / 2M + 1 = 1006]. ¹ H-NMR (methanol-d ₄ ) δ: 8.39 (d, J = 4.7 Hz, 1H), 7.98 (s, 3H), 7.83-7.87 (m, 1H), 7 .77-7.83 (m, 1H), 7.21 (t, J = 5.9 Hz, 1H), 5.23 (d, J = 1.6 Hz, 3H), 4.50-4.64 ( m, 12H), 4.29 (d, J = 5.9 Hz, 3H), 4.16 (t, J = 6.4 Hz, 3H), 3.87-3.96 (m, 12H), 84 (d, J = 7.8 Hz, 3H), 3.71-3.79 (m, 15H), 3.54-3.70 (m, 31H), 3.18-3.28 (m, 2H) ), 3.13 (q, J = 6.5 Hz, 2H), 2.82 (t, J = 7.2 Hz, 2H), 2.12-2.23 (m, 4H), 1.98 (s) 9H), 1.71 (quin, J = 7.3 Hz, 2H), 1.51-1.64 (m, 6H), 1.44-1.51 (m, 11H), 1.28-1. 34 (m, 11H)

N- {6-[(1,3-bis [(1- {1-[(1S, 2R, 3R, 4R, 5S) -4- (acetylamino) -2,3-dihydroxy-6,8-di Oxabicyclo [3.2.1] oct-1-yl] -2,5,8,11-tetraoxatridecan-13-yl} -1H-1,2,3-triazol-4-yl) methoxy] -2-{[(1- {1-[(1S, 2R, 3R, 4R, 5S) -4- (acetylamino) -2,3-dihydroxy-6,8-dioxabicyclo [3.2.1 ] Oct-1-yl] -2,5,8,11-tetraoxatridecan-13-yl} -1H-1,2,3-triazol-4-yl) methoxy] methyl} propan-2-yl) Amino] -6-oxohexyl} -6- (pyridin-2-yldisulfanyl) hexane Bromide (54)
N- {6-[(1,3-bis [(1- {1-[(1S, 2R, 6R, 7R, 8S) -7] in acetic acid (4 mL), methanol (1 mL) and water (1 mL). -(Acetylamino) -4,4-dimethyl-3,5,9,11-tetraoxatricyclo [6.2.1.0-2,6-]-undec-1-yl] -2,5,8 , 11-tetraoxatridecan-13-yl} -1H-1,2,3-triazol-4-yl) methoxy] -2-{[(1- {1-[(1S, 2R, 6R, 7R, 8S) -7- (acetylamino) -4,4-dimethyl-3,5,9,11-tetraoxatricyclo [6.2.1.0-2,6-] undec-1-yl] -2 , 5,8,11-tetraoxatridecan-13-yl} -1H-1,2,3-triazol-4-yl) meth Ci] methyl} propan-2-yl) amino] -6-oxohexyl} -6- (pyridin-2-yldisulfanyl) hexanamide (1-ag-2) (59 mg, 0.029 mmol) Heat at 24 ° C. for 24 hours. After 24 hours, the reaction was cooled to room temperature and concentrated under reduced pressure. The crude material was diluted with toluene and concentrated under reduced pressure. The crude material was diluted a second time with toluene and concentrated under reduced pressure to give the crude title compound (50.5 mg, 91%). The crude material was purified by reverse phase chromatography using the following conditions to give the title compound as a gum (25.2 mg, 45%).

Purification conditions:
The residue was dissolved in dimethyl sulfoxide (1 mL) and purified by reverse phase HPLC. Column: Waters Sunfire C18, 19 × 100, 5u. Mobile phase A: 0.05% TFA in water (v / v). Mobile phase B: 0.05% TFA in acetonitrile (v / v). Gradient: From 80.0% H ₂ O / 20.0% acetonitrile in 10.5 minutes to 70% H ₂ O / 30% acetonitrile in a straight line to 0% H ₂ O / 100% MeCN up to 11.0 minutes, Maintain 0% H ₂ O / 100% acetonitrile from 11.0 to 12.0 minutes. Flow rate: 25 mL / min.

QC condition:
Column: Waters Atlantis dC18, 4.6 × 50, 5u. Mobile phase A: 0.05% TFA in water (v / v). Mobile phase B: 0.05% TFA in acetonitrile (v / v). From 95.0% H ₂ O / 5.0% acetonitrile in 4.0 minutes to 5% H ₂ O / 95% acetonitrile in a straight line 5% H ₂ O / 95% acetonitrile from 4.0 minutes to 5.0 minutes Maintain. Flow rate: 2 mL / min. Retention time = 1.96 minutes. Observed mass = 9466.5137.
Method C: MassLynx \ Acid_3.0 Min. olp-LRMS [1 / 2M + 1 = 946]. ¹ H-NMR (methanol-d ₄ ) δ: 8.45 (d, J = 5.1 Hz, 1H), 8.01 (s, 3H), 7.94 (d, J = 3.1 Hz, 2H) , 7.29-7.36 (m, 1H), 5.21 (s, 3H), 4.57-4.62 (m, 6H), 4.57 (s, 6H), 3.92-4 .00 (m, 6H), 3.89 (dd, J = 10.7, 4.9 Hz, 9H), 3.74-3.80 (m, 9H), 3.71 (dd, J = 10. 1, 4.3 Hz, 3H), 3.53-3.68 (m, 42H), 3.13 (t, J = 6.8 Hz, 2H), 2.85 (t, J = 7.2 Hz, 2H) ), 2.17 (t, J = 7.2 Hz, H), 1.99 (s, 9H), 1.71 (quin, J = 7.4 Hz, 2H), 1.52-1.64 (m , 4H), 1.45 (td, J = 15.0, 7.8 Hz, 4H), 1.26-1.37 (m, 2H)

2- (Pyridin-2-yldisulfanyl) ethyl {6-[(1,3-bis [(1- {1-[(1S, 2R, 6R, 7R, 8S) -7- (acetylamino) -4, 4-Dimethyl-3,5,9,11-tetraoxatricyclo [6.2.1.0-2,6-]-undec-1-yl] -2,5,8,11-tetraoxatridecane- 13-yl} -1H-1,2,3-triazol-4-yl) methoxy] -2-{[(1- {1-[(1S, 2R, 6R, 7R, 8S) -7- (acetylamino) ) -4,4-dimethyl-3,5,9,11-tetraoxatricyclo [6.2.1.0-2,6-]-undec-1-yl] -2,5,8,11-tetra Oxatridecan-13-yl} -1H-1,2,3-triazol-4-yl) methoxy] methyl} Pan 2-yl) amino] -6-oxo-hexyl} carbamate (1-ag-3)
6-Amino-N- (1,3-bis [(1- {1-[(1S, 2R, 6R, 7R,) in N, N-dimethylformamide (0.3 mL) and tetrahydrofuran (0.3 mL). 8S) -7- (acetylamino) -4,4-dimethyl-3,5,9,11-tetraoxatricyclo [6.2.1.0-2,6-] undec-1-yl] -2 , 5,8,11-tetraoxatridecan-13-yl} -1H-1,2,3-triazol-4-yl) methoxy] -2-{[(1- {1-[(1S, 2R, 6R, 7R, 8S) -7- (Acetylamino) -4,4-dimethyl-3,5,9,11-tetraoxatricyclo [6.2.1.0-2,6-] undec-1- Yl] -2,5,8,11-tetraoxatridecan-13-yl} -1H-1,2,3 To a solution of triazol-4-yl) methoxy] methyl} propan-2-yl) hexanamide (1-ag-1) (61.4 mg, 0.0347 mmol) was added N, N-diisopropylethylamine (0.0241 mL, 0 .139 mmol) and 4-nitrophenyl 2- (pyridin-2-yldisulfanyl) ethyl carbonate
(European Journal of Medicinal Chemistry, 82, 355-362, 2014, 18.0 mg, 0.051 mmol) was added for 16 hours at room temperature. After 16 hours, the reaction mixture was concentrated under reduced pressure. The crude material was purified using CombiFlash Rf (RediSep 4 g Gold silica gel column) eluting with a methanol / dichloromethane gradient from 0% to 20% to give the title compound as a gum (57. 4 mg, none, 83%). Method C: MassLynx \ Acid_3.0 Min. olp-LRMS [1 / 2M + 1 = 993]. ¹ H-NMR (methanol-d ₄ ) δ: 8.40 (d, J = 4.3 Hz, 1H), 7.98 (s, 3H), 7.83-7.89 (m, 1H), 7 .75-7.83 (m, 1H), 7.15-7.25 (m, 1H), 5.22 (d, J = 1.2 Hz, 3H), 4.51-4.64 (m, 12H), 4.29 (d, J = 5.9 Hz, 3H), 4.23 (t, J = 6.2 Hz, 2H), 4.15 (t, J = 6.4 Hz, 3H) 3.86 -3.77 (m, 12H), 3.83 (d, J = 7.8 Hz, 3H), 3.72-3.79 (m, 12H), 3.53-3.69 (m, 36H) , 3.05 (t, J = 5.7 Hz, 4H), 2.17 (t, J = 7.0 Hz, 2H), 1.98 (s, 9H), 1.51-1.61 (m, 2 H), 1.48 (s, 11H), 1.33 (s, 11H)

2- (pyridin-2-yldisulfanyl) ethyl {6-[(1,3-bis [(1- {1-[(1S, 2R, 3R, 4R, 5S) -4- (acetylamino) -2, 3-dihydroxy-6,8-dioxabicyclo [3.2.1] oct-1-yl] -2,5,8,11-tetraoxatridecan-13-yl} -1H-1,2,3 -Triazol-4-yl) methoxy] -2-{[(1- {1-[(1S, 2R, 3R, 4R, 5S) -4- (acetylamino) -2,3-dihydroxy-6,8- Dioxabicyclo [3.2.1] oct-1-yl] -2,5,8,11-tetraoxatridecan-13-yl} -1H-1,2,3-triazol-4-yl) methoxy ] Methyl} propan-2-yl) amino] -6-oxohexyl} carbame Doo (55)
2- (Pyridin-2-yldisulfanyl) ethyl {6-[(1,3-bis [(1- {1] in acetic acid (4.0 mL), methanol (1.0 mL) and water (1.0 mL). -[(1S, 2R, 6R, 7R, 8S) -7- (acetylamino) -4,4-dimethyl-3,5,9,11-tetraoxatricyclo [6.2.1.0-2 6-] undec-1-yl] -2,5,8,11-tetraoxatridecan-13-yl} -1H-1,2,3-triazol-4-yl) methoxy] -2-{[( 1- {1-[(1S, 2R, 6R, 7R, 8S) -7- (acetylamino) -4,4-dimethyl-3,5,9,11-tetraoxatricyclo [6.2.1. 0-2,6-] undec-1-yl] -2,5,8,11-tetraoxatridecan-13- L} -1H-1,2,3-triazol-4-yl) methoxy] methyl} propan-2-yl) amino] -6-oxohexyl} carbamate (1-ag-3) (57.4 mg; 0289 mmol) was heated to 70 ° C. for 24 hours. After 24 hours, the reaction was cooled to room temperature and concentrated under reduced pressure. The crude material was diluted with toluene and concentrated under reduced pressure. The crude material was purified by reverse phase chromatography using the following conditions to give the title compound as a gum (29.8 mg, 55%).

Purification conditions:
The residue was dissolved in dimethyl sulfoxide (1 mL) and purified by reverse phase HPLC. Column: Waters Sunfire C18, 19 × 100, 5u. Mobile phase A: 0.05% TFA in water (v / v). Mobile phase B: 0.05% TFA in acetonitrile (v / v). Gradient: From 80.0% H ₂ O / 20.0% acetonitrile in 10.5 minutes to 70% H ₂ O / 30% acetonitrile in a straight line to 0% H ₂ O / 100% MeCN up to 11.0 minutes, Maintain 0% H ₂ O / 100% acetonitrile from 11.0 to 12.0 minutes. Flow rate: 25 mL / min.

QC condition:
Column: Waters Atlantis dC18, 4.6 × 50, 5u. Mobile phase A: 0.05% TFA in water (v / v). Mobile phase B: 0.05% TFA in acetonitrile (v / v). From 95.0% H ₂ O / 5.0% acetonitrile in 4.0 minutes to 5% H ₂ O / 95% acetonitrile in a straight line 5% H ₂ O / 95% acetonitrile from 4.0 minutes to 5.0 minutes Maintain. Flow rate: 2 mL / min. Retention time = 1.91 minutes. Observed mass = 933.4313. Method C: MassLynx \ Acid_3.0 Min. olp-LRMS [1 / 2M + 1 = 933]. ¹ H-NMR (methanol-d ₄ ) δ: 8.46 (d, J = 4.7 Hz, 1H), 8.01 (s, 3H), 7.86-7.97 (m, 2H), 7 .32 (t, J = 5.3 Hz, 1H), 5.21 (s, 3H), 4.55-4.62 (m, 12H), 4.24 (t, J = 6.0 Hz, 2H) , 3.92-3.99 (m, 6H), 3.85-3.92 (m, 9H), 3.74-3.79 (s, 3H), 3.71 (dd, J = 9. 8, 4.3 Hz, 3H), 3.52-3.68 (m, 42H), 2.99-3.15 (m, 4H), 2.17 (t, J = 7.2 Hz, 2H), 1.99 (s, 9H), 1.56 (quin, J = 7.4 Hz, 2H), 1.42-1.50 (m, 2H), 1.24-1.38 (m, 2H)

1-{[6- (4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl) hexanoyl] oxy} pyrrolidine-2,5 (1-ah-1)
A solution of chlorotris (triphenylphosphine) rhodium (I), Wilkinson's catalyst (39.7 mg, 0.0429 mmol) in dichloromethane (5.0 mL) was purged with nitrogen for 10 minutes before 4, 4, 5, 5- Tetramethyl-1,3,2-dioxaborolane (299 mg, 2.34 mmol, 0.340 mL) was added dropwise. The reaction was stirred at room temperature for 10 minutes. 2,5-Dioxopyrrolidin-1-ylhexa-5-enoate
(Journal of the American Chemical Society, 132 (35), 12197-12199, 2010, 412 mg, 1.95 mmol) was dissolved in dichloromethane (1.0 mL) and added dropwise. The reaction was stirred at room temperature for 18 hours. The next morning, the reaction was diluted with dichloromethane and washed with water. The organic layer was dried over magnesium sulfate, filtered and concentrated under reduced pressure. The crude material was purified using CombiFlash Rf (RediSep 24 g Gold silica gel column) eluting with an ethyl acetate / heptane gradient from 0% to 100% to give the crude title compound (366 mg). The crude title compound was purified using CombiFlash Rf (RediSep 24g Gold silica gel column) eluting with a 0% to 100% ethyl acetate / heptane gradient to give the title compound as an oil. (271.0 mg, none, 41.0%). ¹ H-NMR (methanol-d ₄ ) δ: 2.83 (s, 4H), 2.61 (t, J = 7.4 Hz, 2H), 1.71 (quin, J = 7.1 Hz, 2H) , 1.38-1.50 (m, 4H), 1.24 (s, 12H), 0.75 (t, J = 6.8 Hz, 2H).

N- {6-[(1,3-bis [(1- {1-[(1S, 2R, 6R, 7R, 8S) -7- (acetylamino) -4,4-dimethyl-3,5,9 , 11-tetraoxatricyclo [6.2.1.0-2,6-] undec-1-yl] -2,5,8,11-tetraoxatridecan-13-yl} -1H-1, 2,3-triazol-4-yl) methoxy] -2-{[(1- {1-[(1S, 2R, 6R, 7R, 8S) -7- (acetylamino) -4,4-dimethyl-3 , 5,9,11-tetraoxatricyclo [6.2.1.0-2,6-] undec-1-yl] -2,5,8,11-tetraoxatridecan-13-yl}- 1H-1,2,3-triazol-4-yl) methoxy] methyl} propan-2-yl) amino] -6-oxohex -6- (4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl) hexanamide (1-ag-4)
6-amino-N- (1,3-bis [(1- {1-[(1S, 2R, 6R, 7R,) in N, N-dimethylformamide (0.6 mL) and tetrahydrofuran (0.6 mL). 8S) -7- (acetylamino) -4,4-dimethyl-3,5,9,11-tetraoxatricyclo [6.2.1.0-2,6-] undec-1-yl] -2 , 5,8,11-tetraoxatridecan-13-yl} -1H-1,2,3-triazol-4-yl) methoxy] -2-{[(1- {1-[(1S, 2R, 6R, 7R, 8S) -7- (acetylamino) -4,4-dimethyl-3,5,9,11-tetraoxatricyclo [6.2.1.0-2,6-] undec-1- Yl] -2,5,8,11-tetraoxatridecan-13-yl} -1H-1,2,3 To a solution of triazol-4-yl) methoxy] methyl} propan-2-yl) hexanamide (1-ag-1) (200 mg, 0.113 mmol) was added N, N-diisopropylethylamine (0.0786 mL, 0.451 mmol). ) Followed by 1-{[6- (4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl) hexanoyl] oxy} pyrrolidine-2,5-dione (1- ah-1) (57.4 mg, 0.169 mmol) was added and the reaction was stirred at room temperature for 24 hours. After 24 hours, the reaction was concentrated under reduced pressure. The crude material was purified using a CombiFlash Rf (RediSep 12 g Gold silica gel column) eluting with a methanol / dichloromethane gradient from 0% to 20% to give the title compound as a gum ( 209.0 mg, none, 93%). Method C: LRMS operating for 3 minutes [1 / 2M = 998]. ¹ H-NMR (methanol-d ₄ ) δ: 7.98 (s, 3H), 5.23 (d, J = 1.6 Hz, 3H), 4.52-4.62 (m, 12H), 4 .29 (d, J = 5.9 Hz, 3H), 4.16 (t, J = 6.4 Hz, 3H), 3.87-3.97 (m, 12H), 3.84 (d, J = 8.2 Hz, 3H), 3.72-3.79 (m, 12H), 3.54-3.69 (m, 36H), 3.13 (q, J = 6.6 Hz, 2H), 16 (q, J = 7.3 Hz, 4H), 1.98 (s, 9H), 1.52-1.66 (m, 4H), 1.44-1.51 (m, 11H), 35-1.43 (m, 2H), 1.27-1.35 (m, 13H), 1.18-1.25 (m, 12H), 0.73 (t, J = 7.6 Hz, 2H)

N- {6 [(1,3-bis [(1- {1-[(1S, 2R, 3R, 4R, 5S) -4- (acetylamino) -2,3-dihydroxy-6,8-dioxa Bicyclo [3.2.1] oct-1-yl] -2,5,8,11-tetraoxatridecan-13-yl} -1H-1,2,3-triazol-4-yl) methoxy]- 2-{[(1- {1-[(1S, 2R, 3R, 4R, 5S) -4- (acetylamino) -2,3-dihydroxy-6,8-dioxabicyclo [3.2.1] Oct-1-yl] -2,5,8,11-tetraoxatridecan-13-yl} -1H-1,2,3-triazol-4-yl) methoxy] methyl} propan-2-yl) amino ] -6-oxohexyl} -6- (4,4,5,5-tetramethyl-1,3,2-di Kisabororan 2-yl) hexanamide (56)
N- {6-[(1,3-bis [(1- {1-[(1S, 2R, 6R, 7R, 8S) -7] in acetic acid (4 mL), methanol (1 mL) and water (1 mL). -(Acetylamino) -4,4-dimethyl-3,5,9,11-tetraoxatricyclo [6.2.1.0-2,6-]-undec-1-yl] -2,5,8 , 11-tetraoxatridecan-13-yl} -1H-1,2,3-triazol-4-yl) methoxy] -2-{[(1- {1-[(1S, 2R, 6R, 7R, 8S) -7- (acetylamino) -4,4-dimethyl-3,5,9,11-tetraoxatricyclo [6.2.1.0-2,6-] undec-1-yl] -2 , 5,8,11-tetraoxatridecan-13-yl} -1H-1,2,3-triazol-4-yl) meth Ci] methyl} propan-2-yl) amino] -6-oxohexyl-6- (4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl) hexanamide (1-ag -4) A solution of (104.0 mg, 0.0521 mmol) was heated at 70 ° C. for 24 hours. After 24 hours, the reaction was cooled to room temperature and concentrated under reduced pressure. The crude material was diluted with toluene and concentrated under reduced pressure. The crude material was diluted a second time with toluene and concentrated under reduced pressure to give the crude title compound (112.0 mg, 115%). A portion of this crude title compound (52.7 mg) was purified by reverse phase chromatography to give the title compound as a gum (18.2 mg, 19%).

Purification conditions The residue was dissolved in dimethyl sulfoxide (1 mL) and purified by reverse phase HPLC. Column: Waters Sunfire C18 19 × 100 · 5u. Mobile phase A: 0.05% TFA in water (v / v). Mobile phase B: 0.05% TFA in acetonitrile (v / v). Gradient: 80.0% H ₂ O / 20.0% acetonitrile in 8.5 minutes to 65% H ₂ O / 35% acetonitrile in a straight line 9, 0% H ₂ O / 100% MeCN, 9 to 0 minutes, 9 Maintain 0% H ₂ O / 100% acetonitrile from 0 to 10.0 minutes. Flow rate: 25 mL / min.

QC conditions Column: Waters Atlantis dC18, 4.6 × 50, 5u. Mobile phase A: 0.05% TFA in water (v / v). Mobile phase B: 0.05% TFA in acetonitrile (v / v). Gradient: 95.0% H ₂ O / 5.0% acetonitrile at 4.0 minutes to 5% H ₂ O / 95% acetonitrile linearly from 4.0 minutes to 5.0% 5% H ₂ O / 95 % Acetonitrile maintained. Flow rate: 2 mL / min. Retention time = 2 minutes. Observed mass = 938.9628. ¹ H-NMR (methanol-d ₄ ) δ: 8.01 (s, 3H), 5.21 (s, 3H), 4.51-4.66 (m, 12H), 3.95 (dd, J = 9.4, 5.9 Hz, 6H), 3.89 (dd, J = 11.7, 4.7 Hz, 9H), 3.74-3.81 (m, 9H), 3.71 (dd, J = 9.8, 4.3 Hz, 3H), 3.52-3.68 (m, 42H), 3.13 (t, J = 2H), 2.17 (q, J = 7.0 Hz, 4H) ), 1.99 (s, 9H), 1.53-1.66 (m, 4H), 1.45-1.52 (m, 2H), 1.36-1.44 (m, 2H), 1.27-1.35 (m, 4H), 1.23 (s, 12H), 0.73 (t, J = 7.6 Hz, 2H)

Ethyl 7-[(1,3-dihydroxypropan-2-yl) amino] -7-oxoheptanoate (1-ai-1)
Ethyl 7-[(2,5-dioxopyrrolidin-1-yl) oxy] -7-oxoheptanoate (1-z-1) (228.0 mg, N, N-dimethylformamide (1.0 mL) N, N-diisopropylethylamine (0.557 mL, 3.20 mmol) was added to a solution of 0.799 mmol), and the mixture was stirred for 10 minutes, and then 2-aminopropane-1,3-diol (72.8 mg, 0.7. 799 mmol) was added and the reaction was stirred at room temperature for 72 hours. After 72 hours, the reaction was diluted with water and extracted three times with dichloromethane. The combined organic layers were washed with brine, dried over magnesium sulfate, filtered and concentrated under reduced pressure to give the crude title compound (89.0 mg, none, 43%). The aqueous layer was concentrated under reduced pressure. The crude concentrated aqueous layer was diluted with methanol (5 mL) and dichloromethane (10 mL). The mixture was decanted and combined with the crude title compound from the first extraction. The solution was concentrated under reduced pressure. The combined crude material was purified using CombiFlash Rf (RediSep 12 g silica gel column) eluting with a methanol / dichloromethane gradient from 0% to 20% to give the title compound (183.0 mg, 88%). Method C: 3-minute operation LRMS [M + 1 = 262]. ¹ H-NMR (methanol-d ₄ ) δ: 4.11 (q, J = 7.2 Hz, 2H), 3.83-3.99 (m, 1H), 3.60 (d, J = 5. 5 Hz, 4H), 2.31 (t, J = 7.2 Hz, 2H), 2.33 (t, J = 7.4 Hz, 2H), 1.63 (quin, J = 7.5 Hz, 4H), 1.30-1.45 (m, 2H), 1.24 (t, J = 7.0 Hz, 3H)

Ethyl 7-[(2,2-dimethyl-1,3-dioxane-5-yl) amino] -7-oxoheptanoate (1-aj-1)
Ethyl 7-[(1,3-dihydroxypropan-2-yl) amino] -7-oxoheptanoate (1-ai-1) (180.0 mg, 0.689 mmol) in N, N-dimethylformamide (2 mL) ) Was added 2,2-dimethoxypropane (0.53 mL, 4.13 mmol) followed by (1S)-(+)-10-camphorsulfonic acid (64.0 mg, 0.276 mmol). . The reaction was heated to 70 ° C. for 72 hours. After 72 hours, the reaction was cooled to room temperature and partitioned between water (20 mL) and ethyl acetate (10 mL). The layers were extracted and the layers were separated. The aqueous layer was washed twice more with ethyl acetate (10 mL). The combined organic layers were washed with water, brine, dried over sodium sulfate, filtered and concentrated under reduced pressure to give the crude title compound (94.0 mg, none, 45%).

7-[(2,2-Dimethyl-1,3-dioxane-5-yl) amino] -7-oxoheptanoic acid (1-ak-1)
Ethyl 7-[(2,2-dimethyl-1,3-dioxan-5-yl) amino] -7-oxoheptanoate (1-aj-1) (94.0 mg, 0.31 mmol) in ethanol (5 mL) ) Was added 1.0 M aqueous sodium hydroxide (1.5 mL, 1.5 mmol) and the reaction was stirred at room temperature overnight. The next morning, the reaction was concentrated under reduced pressure. The resulting crude material was diluted with 1N hydrochloric acid (3.0 mL) and ethyl acetate. The layers were separated and the organic layer was extracted twice more with ethyl acetate. The combined organic layers were washed with brine, dried over sodium sulfate, filtered and concentrated under reduced pressure to give the crude title compound (29.4 mg, none, 34%).

N- {6 [(1,3-bis [(1- {1-[(1S, 2R, 3R, 4R, 5S) -4- (acetylamino) -2,3-dihydroxy-6,8-dioxa Bicyclo [3.2.1] oct-1-yl] -2,5,8,11-tetraoxatridecan-13-yl} -1H-1,2,3-triazol-4-yl) methoxy]- 2-{[(1- {1-[(1S, 2R, 3R, 4R, 5S) -4- (acetylamino) -2,3-dihydroxy-6,8-dioxabicyclo [3.2.1] Oct-1-yl] -2,5,8,11-tetraoxatridecan-13-yl} -1H-1,2,3-triazol-4-yl) methoxy] methyl} propan-2-yl) amino ] -6-oxohexyl} -N ′-(1,3-dihydroxypropan-2-yl) hept Tandiamide (57)
7-[(2,2-Dimethyl-1,3-dioxan-5-yl) amino] -7-oxoheptanoic acid (N, N-dimethylformamide (0.3 mL) and tetrahydrofuran (0.3 mL) 1-ak-1) (18.8 mg, 0.0688 mmol) in 1-hydroxybenzotriazole (10.3 mg, 0.0762 mmol) and N- (3-dimethylaminopropyl) -N′-ethylcarbodiimide hydrochloride Salt (14.9 mg, 0.0762 mmol) was added and the reaction was stirred at room temperature for 1 hour. This reaction mixture was reacted with 6-amino-N- (1,3-bis [(1- {1-[(1S, 2R, 6R, 7R, 8S) -7- (acetylamino) -4,4-dimethyl- 3,5,9,11-tetraoxatricyclo [6.2.1.0-2,6-] undec-1-yl] -2,5,8,11-tetraoxatridecan-13-yl} -1H-1,2,3-triazol-4-yl) methoxy] -2-{[(1- {1-[(1S, 2R, 6R, 7R, 8S) -7- (acetylamino) -4, 4-Dimethyl-3,5,9,11-tetraoxatricyclo [6.2.1.0-2,6-]-undec-1-yl] -2,5,8,11-tetraoxatridecane- 13-yl} -1H-1,2,3-triazol-4-yl) methoxy] methyl} propan-2-yl) he Sanamide (1-ag-1) (75.0 mg, 0.042 mmol) was added followed by N, N-diisopropylethylamine (0.0295 mL, 0.169 mmol) and the reaction was stirred at room temperature for 16 hours. . After 16 hours, the reaction was concentrated under reduced pressure. This crude material was dissolved in acetic acid (4.0 mL), methanol (1 mL) and water (1.0 mL) and heated to 70 ° C. for 24 hours. After 24 hours, the reaction was cooled to room temperature and concentrated under reduced pressure. The crude material was diluted with toluene and concentrated under reduced pressure to give the crude title compound (175.0 mg, 220%). The title compound was purified by reverse phase chromatography using the following conditions to give the title compound as a gum (10.9 mg, 14%).

Purification conditions:
The residue was dissolved in dimethyl sulfoxide (1 mL) and purified by reverse phase HPLC. Column: Waters Sunfire C18, 19 × 100, 5u. Mobile phase A: 0.05% TFA in water (v / v). Mobile phase B: 0.05% TFA in acetonitrile (v / v). Gradient: 85.0% H ₂ O / 15.0% acetonitrile in 8.5 minutes linearly to 75% H ₂ O / 25% acetonitrile, 0% H ₂ O / 100% MeCN up to 9.0 minutes, Maintain 0% H ₂ O / 100% acetonitrile from 9.0 minutes to 10.0 minutes. Flow rate: 25 mL / min.

QC condition:
Column: Waters Atlantis dC18, 4.6 × 50, 5u. Mobile phase A: 0.05% TFA in water (v / v). Mobile phase B: 0.05% TFA in acetonitrile (v / v). From 95.0% H ₂ O / 5.0% acetonitrile in 4.0 minutes to 5% H ₂ O / 95% acetonitrile in a straight line 5% H ₂ O / 95% acetonitrile from 4.0 minutes to 5.0 minutes Maintain. Flow rate: 2 mL / min. Retention time = 1.53 minutes. Observed mass = 934.548. Method C: LRMS [M + Na = 1890] running for 3 minutes. ¹ H-NMR (methanol-d ₄ ) δ: 8.00 (s, 3H), 5.21 (s, 3H), 4.52-4.62 (m, 12H), 3.95 (t, J = 9.4 Hz, 6H), 3.85-3.91 (m, 9H), 3.74-3.79 (m, 9H), 3.71 (dd, J = 10.0, 4.1 Hz, 3H), 3.55-3.67 (m, 47H), 3.12 (t, J = 6.7 Hz, 2H), 2.22 (t, J = 7.3 Hz, 2H), 2.17 ( t, J = 7.3 Hz, 4H), 1.98 (s, 9H), 1.58-1.69 (m, 4H), 1.51-1.57 (m, 2H), 1.48 ( quin, J = 7.2 Hz, 2H), 1.26-1.40 (m, 4H)

6-azido-N- {6-[(1,3-bis [(1- {1 [(1S, 2R, 6R, 7R, 8S) -7- (acetylamino) -4,4-dimethyl-3, 5,9,11-tetraoxatricyclo [6.2.1.0-2,6-] undec-1-yl] -2,5,8,11-tetraoxatridecan-13-yl} -1H -1,2,3-triazol-4-yl) methoxy] -2-{[(1- {1-[(1S, 2R, 6R, 7R, 8S) -7- (acetylamino) -4,4- Dimethyl-3,5,9,11-tetraoxatricyclo [6.2.1.0-2,6-]-undec-1-yl] -2,5,8,11-tetraoxatridecan-13 Yl} -1H-1,2,3-triazol-4-yl) methoxy] methyl} propan-2-yl) -6-oxohe Sill} hexanamide (1-ag-5)
6-amino-N- (1,3-bis [(1- {1-[(1S, 2R, 6R, 7R,) in N, N-dimethylformamide (0.6 mL) and tetrahydrofuran (0.6 mL). 8S) -7- (acetylamino) -4,4-dimethyl-3,5,9,11-tetraoxatricyclo [6.2.1.0-2,6-] undec-1-yl] -2 , 5,8,11-tetraoxatridecan-13-yl} -1H-1,2,3-triazol-4-yl) methoxy] -2-{[(1- {1-[(1S, 2R, 6R, 7R, 8S) -7- (acetylamino) -4,4-dimethyl-3,5,9,11-tetraoxatricyclo [6.2.1.0-2,6-] undec-1- Yl] -2,5,8,11-tetraoxatridecan-13-yl} -1H-1,2,3 To a solution of triazol-4-yl) methoxy] methyl} propan-2-yl) hexanamide (1-ag-1) (300 mg, 0.169 mmol) was added N, N-diisopropylethylamine (0.118 mL, 0.677 mmol). ) And 1-[(6-azidohexanoyl) oxy] pyrrolidine-2,5-dione (56.0 mg, 0.220 mmol) were added. The reaction was stirred at room temperature for 24 hours. After 24 hours, the reaction was concentrated under reduced pressure. The crude reaction mixture was purified using CombiFlash Rf (RediSep 24 g Gold silica gel column) eluting with a methanol / dichloromethane gradient from 0% to 20% to give the title compound as a gum ( 269 mg, 83%). Method C: 3-minute run LRMS [1 / 2M + 1 = 956]. ¹ H-NMR (methanol-d ₄ ) δ: 7.98 (s, 3H), 5.22 (s, 3H), 4.50-4.65 (m, 12H), 4.29 (d, J = 5.9 Hz, 3H), 4.16 (t, J = 6.5 Hz, 3H), 3.83-3.95 (m, 12H), 3.83 (d, J = 7.6 Hz, 3H) , 3.73-3.79 (m, 12H), 3.55-3.71 (m, 36H), 3.26-3.30 (m, 2H), 3.14 (q, J = 6. 5Hz, 2H), 2.18 (q, J = 7.6 Hz, 4H), 1.98 (s, 9H), 1.53-1.68 (m, 6H), 1.45-1.51 ( m, 11H), 1.36-1.43 (m, 2H), 1.29-1.36 (m, 11H)

6-azido-N- {6- (1,3-bis [1- {1-[(1S, 2R, 3R, 4R, 5S) -4- (acetylamino) -2,3-dihydroxy-6,8 -Dioxabicyclo [3.2.1] oct-1-yl] -2,5,8,11-tetraoxatridecan-13-yl} -1H-1,2,3-triazol-4-yl) Methoxy] 2-{[(1- {1-[(1S, 2R, 3R, 4R, 5S) -4- (acetylamino) -2,3-dihydroxy-6,8-dioxabicyclo [3.2. 1] Oct-1-yl] -2,5,8,11-tetraoxatridecan-13-yl} -1H-1,2,3-triazol-4-yl) methoxy] methyl} propan-2-yl Amino] -6-oxohexyl} hexanamide (58)
6-azido-N- {6-[(1,3-bis [(1- {1 [(1S, 2R, 6R) in acetic acid (3 mL), methanol (0.75 mL) and water (0.75 mL). , 7R, 8S) -7- (acetylamino) -4,4-dimethyl-3,5,9,11-tetraoxatricyclo [6.2.1.0-2,6-] undec-1-yl ] -2,5,8,11-tetraoxatridecan-13-yl} -1H-1,2,3-triazol-4-yl) methoxy] -2-{[(1- {1-[(1S , 2R, 6R, 7R, 8S) -7- (acetylamino) -4,4-dimethyl-3,5,9,11-tetraoxatricyclo [6.2.1.0-2,6-] undeca -1-yl] -2,5,8,11-tetraoxatridecan-13-yl} -1H-1,2,3-tria Ol-4-yl) methoxy] methyl} propan-2-yl) -6-oxohexyl} hexanamide (1-ag-5) (25.0 mg, 0.013 mmol) was stirred at 70 ° C. for 24 hours. Heated. After 24 hours, the reaction was cooled to room temperature and concentrated under reduced pressure. The crude material was diluted with toluene and concentrated under reduced pressure to give the title compound as a gum (22.7 mg, 97%). Method C: 3-minute operation LRMS [M + 1 = 1791]. ¹ H-NMR (methanol-d ₄ ) δ: 7.9 (s, 3H), 5.21 (s, 3H), 4.51-4.66 (m, 12H), 3.92-4.01 (M, 6H), 3.89 (dd, J = 4.7 Hz, 9H), 3.74-3.81 (m, 9H), 3.71 (dd, J = 10.0, 4.1 Hz, 3H), 3.52-3.68 (m, 42H), 3.25-3.30 (m, 2H), 3.08-3.19 (m, 2H), 2.13-2.23 ( m, 4H), 1.99 (s, 9H), 1.54-1.69 (m, 6H), 1.49 (dt, J = 14.4, 7.2 Hz, 2H), 1.36 1.44 (m, 2H), 1.32 (dd, J = 14.8, 6.2 Hz, 2H)

1-{[6- (Benzyloxy) hexanoyl] oxy} pyrrolidine-2,5-dione (1-al-1)
6- (Benzyloxy) hexanoic acid in N, N-dimethylformamide (20 mL)
(Synlett, (4), 693-697, see 2004, 1400.0 mg, 6.298 mmol) was added N-hydroxysuccinimide (870 mg, 7.56 mmol), followed by N- (3-dimethylaminopropyl). ) -N'-ethylcarbodiimide hydrochloride (1480 mg, 7.56 mmol) was added. The reaction was stirred overnight at room temperature. The next morning, the reaction was quenched with water and extracted three times with dichloromethane. The combined organic layers were washed with saturated sodium bicarbonate, brine, dried over sodium sulfate, filtered and concentrated under reduced pressure. Purification of the crude material using a CombiFlash • Rf (RediSep • 40 g • gold column) eluting with an ethyl acetate / heptane gradient from 0% to 100% gave the title compound as a gum (715 mg). 36%). Method C: LRMS [M + Na = 342] running for 1.5 minutes. ¹ H-NMR (methanol-d ₄ ) δ: 7.22-7.42 (m, 5H), 4.51 (s, 2H), 3.53 (t, J = 6.4 Hz, 2H), 2 .85 (s, 4H), 2.65 (t, 7.4 Hz, 2H), 1.76 (quin, J = 7.5 Hz, 2H), 1.61-1.71 (m, 2H), 1 .47-1.59 (m, 2H)

6- (Benzyloxy) -N- {6-[(1,3-bis {1- {1-[[(1S, 2R, 6R, 7R, 8S) -7- (acetylamino) -4,4- Dimethyl-3,5,9,11-tetraoxatricyclo [6.2.1.0-2,6-]-undec-1-yl] -2,5,8,11-tetraoxatridecan-13 Yl} -1H-1,2,3-triazol-4-yl) methoxy] -2-{[(1- {1-[(1S, 2R, 6R, 7R, 8S) -7- (acetylamino)- 4,4-Dimethyl-3,5,9,11-tetraoxatricyclo [6.2.1.0-2,6-]-undec-1-yl] -2,5,8,11-tetraoxatri Decan-13-yl} 1H-1,2,3-triazol-4-yl) methoxy] methyl} propan-2-yl) a Roh] -6-oxo-hexyl} hexanamide (1-ag-6)
6-amino-N- (1,3-bis [(1- {1-[(1S, 2R, 6R, 7R,) in N, N-dimethylformamide (0.6 mL) and tetrahydrofuran (0.6 mL). 8S) -7- (acetylamino) -4,4-dimethyl-3,5,9,11-tetraoxatricyclo [6.2.1.0-2,6-] undec-1-yl] -2 , 5,8,11-tetraoxatridecan-13-yl} -1H-1,2,3-triazol-4-yl) methoxy] -2-{[(1- {1-[(1S, 2R, 6R, 7R, 8S) -7- (acetylamino) -4,4-dimethyl-3,5,9,11-tetraoxatricyclo [6.2.1.0-2,6-] undec-1- Yl] -2,5,8,11-tetraoxatridecan-13-yl} -1H-1,2,3 To a solution of triazol-4-yl) methoxy] methyl} propan-2-yl) hexanamide (1-ag-1) (200 mg, 0.113 mmol) was added N, N-diisopropylethylamine (0.0786 mL, 0.451 mmol). ) And 1-{[6- (benzyloxy) hexanoyl] oxy} pyrrolidine-2,5-dione (1-al-1) (46.9 mg, 0.147 mmol) were added and the reaction was allowed to proceed for 24 hours at room temperature. Stir. After 24 hours, the reaction was concentrated under reduced pressure. The crude material was purified using CombiFlash Rf (RediSep 12 g Gold silica gel column) eluting with a methanol / dichloromethane gradient from 0% to 20% to give the title compound as a gum ( 203 mg, 91%). ¹ H-NMR (methanol-d ₄ ) δ: 7.98 (s, 3H), 7.19-7.38 (m, 5H), 5.23 (d, J = 1.2 Hz, 3H), 4 .52-4.64 (m, 12H), 4.48 (s, 2H), 4.29 (d, J = 5.9 Hz, 3H), 4.15 (t, J = 6.4 Hz, 3H) , 3.86 to 3.96 (m, 12H), 3.83 (d, J = 7.8 Hz, 3H), 3.72 to 3.80 (m, 12H), 3.54-3.70 ( m, 36H), 3.49 (t, J = 6.4 Hz, 2H), 3.08-3.15 (m, 2H), 2.12-2.26 (m, 4H), 1.98 ( s, 9H), 1.51-1.68 (m, 6H), 1.48 (s, 11H), 1.37-1.44 (m, 2H), 1.33 (s, 11H)

6- (Benzyloxy) -N- {6-[(1,3-bis [(1- {1-[(1S, 2R, 3R, 4R, 5S) -4- (acetylamino) -2,3- Dihydroxy-6,8-dioxabicyclo [3.2.1] oct-1-yl] -2,5,8,11-tetraoxatridecan-13-yl} -1H-1,2,3-triazole -4-yl) methoxy) -2-{[(1- {1-[(1S, 2R, 3R, 4R, 5S) -4- (acetylamino) -2,3-dihydroxy-6,8-dioxa Bicyclo [3.2.1] oct-1-yl] -2,5,8,11-tetraoxatridecan-13-yl} -1H-1,2,3-triazol-4-yl) methoxy] methyl } Propan-2-yl) amino] -6-oxohexyl] hexanamide (59)
6- (Benzyloxy) -N- {6-[(1,3-bis {1- {1-[[in acetic acid (6.0 ml) methanol) (1.5 mL) and water (1.5 mL). (1S, 2R, 6R, 7R, 8S) -7- (acetylamino) -4,4-dimethyl-3,5,9,11-tetraoxatricyclo [6.2.1.0-2,6- ] Undec-1-yl] -2,5,8,11-tetraoxatridecan-13-yl} -1H-1,2,3-triazol-4-yl) methoxy] -2-{[(1- {1-[(1S, 2R, 6R, 7R, 8S) -7- (acetylamino) -4,4-dimethyl-3,5,9,11-tetraoxatricyclo [6.2.1.0 2,6-] undec-1-yl] -2,5,8,11-tetraoxatridecan-13-yl} 1H-2,3 A solution of triazol-4-yl) methoxy] methyl} propan-2-yl) amino] -6-oxohexyl} hexanamide (1-ag-6) (180.0 mg, 0.0911 mmol) was heated to 70 ° C. at 24 ° C. Heated for hours. After 24 hours, the reaction was cooled to room temperature and concentrated under reduced pressure. The crude material was diluted with toluene and concentrated under reduced pressure. The crude material was diluted a second time with toluene and concentrated under reduced pressure to give the title compound as a gum (164.0 mg, 97.0%). Method C: LRMS operating for 3 minutes [1 / 2M = 928]. ¹ H-NMR (methanol-d ₄ ) δ: 7.9 (s, 3H), 7.25-7.39 (m, 5H), 5.21 (s, 3H), 4.52-4.66 (M, 12H), 4.48 (s, 2H), 3.92-3.99 (m, 6H), 3.84-3.91 (m, 9H), 3.74-3.81 (m 9H), 3.71 (dd, J = 9.8, 4.3 Hz, 3H), 3.54-3.67 (m, 42H), 3.49 (t, J = 6.4 Hz, 2H) , 3.08-3.17 (m, 2H), 2.13-2.22 (m, 4H), 1.99 (s, 9H), 1.51-1.68 (m, 6H), 1 .48 (t, J = 7.4 Hz, 2H), 1.39 (dt, J = 15.3, 7.8 Hz, 2H), 1.26-1.35 (m, 2H)

(1S, 2R, 3R, 4R, 5S) -4- (acetylamino) -2- (acetyloxy) -1- {13- [4- (4,4-bis {[(1- {1-[( 1S, 2R, 3R, 4R, 5S) -4- (acetylamino) -2,3-bis (acetyloxy) -6,8-dioxabicyclo [3.2.1] oct-1-yl] -2 , 5,8,11-tetraoxatridecan-13-yl} -1H-1,2,3-triazol-4-yl) methoxy] methyl} -6,13-dioxo-20-phenyl-2,19- Dioxa-5,12-diazaicos-1-yl) -1H-1,2,3-triazol-1-yl] -2,5,8,11-tetraoxatridec-1-yl} -6,8- Dioxabicyclo [3.2.1] oct-3-yl acetate (l-am-1)
6- (Benzyloxy) -N- {6-[(1,3-bis [(1- {1-[(1S, 2R, 3R, 4R, 5S) -4- (acetylamino) -2,3- Dihydroxy-6,8-dioxabicyclo [3.2.1] oct-1-yl] -2,5,8,11-tetraoxatridecan-13-yl} -1H-1,2,3-triazole -4-yl) methoxy) -2-{[(1- {1-[(1S, 2R, 3R, 4R, 5S) -4- (acetylamino) -2,3-dihydroxy-6,8-dioxa Bicyclo [3.2.1] oct-1-yl] -2,5,8,11-tetraoxatridecan-13-yl} -1H-1,2,3-triazol-4-yl) methoxy] methyl } Propan-2-yl) amino] -6-oxohexyl] hexanamide (59) (1 0 mg, the 0.07mmol), (3mL, dissolved in 40 mmol), acetic anhydride was added at room temperature (0.198mL, 2.10mmol) thereto. The reaction was then heated to 50 ° C. overnight. The next morning, the reaction was concentrated under reduced pressure. The crude material was purified using a CombiFlash Rf (RediSep 12 g Gold silica gel column) eluting with a methanol / dichloromethane gradient from 0% to 20% to give the title compound as a gum ( 130.0 mg, 88%). Method C: 3-minute operation LRMS [1 / 2M = 1054]. ¹ H-NMR (methanol-d ₄ ) δ: 7.98 (s, 3H), 7.16-7.39 (m, 5H), 5.44 (d, J = 4.3 Hz, 3H), 5 .32 (s, 3H), 5.10 (dd, J = 10.5, 4.3 Hz, 3H), 4.52-4.60 (m, 12H), 4.48 (s, 2H) 18 (d, J = 10.5 Hz, 3H), 3.99 (d, J = 8.2 Hz, 3H), 3.89 (t, J = 5.1 Hz, 6H), 3.70-3.81 (M, 12H), 3.52-3.67 (m, 39H), 3.49 (t, J = 6.2 Hz, 2H), 3.13 (q, J = 6.6 Hz, 2H), 2 .13-2.21 (m, 13H), 1.94 (d, J = 1.6 Hz, 18H), 1.51-1.68 (m, 6H), 1.45-1.50 ( m, 2H), 1.37-1.43 (m, 2H), 1.28-1.35 (m, 2H)

N- {6-[(1,3-bis [(1- {1-[(1S, 2R, 3R, 4R, 5S) -4- (acetylamino) -2,3-dihydroxy-6,8-di Oxabicyclo [3.2.1] oct-1-yl] -2,5,8,11-tetraoxatridecan-13-yl} -1H-1,2,3-triazol-4-yl) methoxy] -2-{[(1- {1-[(1S, 2R, 3R, 4R, 5S) -4- (acetylamino) -2,3-dihydroxy-6,8-dioxabicyclo [3.2.1 ] Oct-1-yl] -2,5,8,11-tetraoxatridecan-13-yl} -1H-1,2,3-triazol-4-yl) methoxy] methyl} propan-2-yl) Amino] -6-oxohexyl} -6-hydroxyhexanamide (1-an-1)
(1S, 2R, 3R, 4R, 5S) -4- (acetylamino) -2- (acetyloxy) -1- {13- [4- (4,4-bis {[(1- {1-[( 1S, 2R, 3R, 4R, 5S) -4- (acetylamino) -2,3-bis (acetyloxy) -6,8-dioxabicyclo [3.2.1] oct-1-yl] -2 , 5,8,11-tetraoxatridecan-13-yl} -1H-1,2,3-triazol-4-yl) methoxy] methyl} -6,13-dioxo-20-phenyl-2,19- Dioxa-5,12-diazaicos-1-yl) -1H-1,2,3-triazol-1-yl] -2,5,8,11-tetraoxatridec-1-yl} -6,8- Dioxabicyclo [3.2.1] oct-3-yl acetate (l-am-1) 110 mg, the 0.0522Mmol) (dissolved in 10.0 mL), then this solution the following parameters (temperature = 60 ° C., flow rate = 1.0 mL / min, pressure = total H ₂ (1 bar) methanol) using And passed through an H-cube using 10% palladium on carbon (small cartridge). The solution was collected and concentrated under reduced pressure to give the title compound as a gum (91.6 mg, 87%). Method C: 3-minute operation LRMS [1 / 2M = 1090]. ¹ H-NMR (methanol-d ₄ ) δ: 7.98 (s, 3H), 5.44 (d, J = 4.3 Hz, 3H), 5.32 (s, 3H), 5.10 (dd , J = 10.5, 4.3 Hz, 3H), 4.50-4.64 (m, 12H), 4.18 (d, J = 10.5 Hz, 3H), 3.99 (d, J = 8.2 Hz, 3H), 3.90 (t, J = 4.9 Hz, 6H), 3.71-3.82 (m, 9H), 3.44-3.66 (m, 44H), 08-3.19 (m, 2H), 2.16-2.22 (m, 4H), 2.15 (s, 9H), 1.94 (d, J = 1.2 Hz, 18H), 44-1.68 (m, 8H), 1.27-1.42 (m, 4H)

(1S, 2R, 3R, 4R, 5S) -4- (acetylamino) -1- {13- [4-({3-[(1- {1-[(1S, 2R, 3R, 4R, 5S) -4- (acetylamino) -2,3-bis (acetyloxy) -6,8-dioxabicyclo [3.2.1] oct-1-yl] -2,5,8,11-tetraoxatri Decan-13-yl} -1H-1,2,3-triazol-4-yl) methoxy] -2-{[(1- {1-[(1S, 2R, 3R, 4R, 5S) -4- ( Acetylamino) -2,3-bis (acetyloxy) -6,8-dioxabicyclo [3.2.1] oct-1-yl] -2,5,8,11-tetraoxatridecan-13 Yl} -1H-1,2,3-triazol-4-yl] methoxy] methyl} -2-({6 [(6-hydroxy Hexanoyl) amino] hexanoyl} amino) propoxy} methyl) -1H-1,2,3-triazol-1-yl] -2,5,8,11-tetraoxatridec-1-yl} -3- (acetyl Oxy) -6,8-dioxabicyclo [3.2.1] oct-2-yl acetate (60)
6- (Benzyloxy) -N- {6-[(1,3-bis [(1- {1-[(1S, 2R, 3R, 4R, 5S) -4- (acetylamino) -2,3- Dihydroxy-6,8-dioxabicyclo [3.2.1] oct-1-yl] -2,5,8,11-tetraoxatridecan-13-yl} -1H-1,2,3-triazole -4-yl) methoxy] -2-{[(1- {1-[(1S, 2R, 3R, 4R, 5S) -4- (acetylamino) -2,3-dihydroxy-6,8-dioxa Bicyclo [3.2.1] oct-1-yl] -2,5,8,11-tetraoxatridecan-13-yl} -1H-1,2,3-triazol-4-yl) methoxy] methyl } Propan-2-yl) amino] -6-oxohexyl} hexanamide (1-an- ) (31.0 mg, and 0.017 mmol) was dissolved in methanol (5 mL), then this solution the following parameters (temperature = 60 ° C., flow rate = 1.0 mL / min, pressure = total H ₂ (1 bar)) Was passed through an H-cube with 10% palladium on carbon (small cartridge). The solution was collected and concentrated under reduced pressure to give the title compound as a gum (7.9 mg, 27%). Method C: 3-minute run LRMS [M + 1 = 1766]. ¹ H-NMR (methanol-d ₄ ) δ: 7.9 (s, 3H), 5.21 (s, 3H), 4.49-4.63 (m, 12H), 3.92-4.00 (M, 6H), 3.89 (dd, J = 3.5 Hz, 9H), 3.74-3.79 (m, 9H), 3.71 (dd, J = 9.8, 4.3 Hz, 3H), 3.53-3.67 (m, 44H), 3.02-3.16 (m, 2H), 2.18 (td, J = 7.3, 3.3 Hz, 4H), 99 (s, 9H), 1.44-1.72 (m, 8H), 1.25-1.42 (m, 4H)

S, 2R, 6R, 7R, 8S) -7- (acetylamino) -4,4-dimethyl-3,5,9,11-tetraoxatricyclo [6.2.1.0-2,6- Undec-1-yl] -2,5,8,11-tetraoxatridecan-13-yl] -1H-1,2,3-triazol-4-yl) methoxy] 2-{[(1- {1 -[1S, 2R, 6R, 7R, 8S) -7- (acetylamino) -4,4-dimethyl-3,5,9,11-tetraoxatricyclo [6.2.1.0-2,6 ~] Undec-1-yl] -2,5,8,11-tetraoxatridecan-13-yl] -1H-1,2,3-triazol-4-yl} methoxy] methyl} propan-2-yl ) Amino] -6-oxohexyl} amino) -6-oxohexyl] carbamate ( -ag-7)
6-amino-N- (1,3-bis [(1- {1-[(1S, 2R, 6R, 7R,) in N, N-dimethylformamide (1.5 mL) and tetrahydrofuran (1.0 mL). 8S) -7- (acetylamino) -4,4-dimethyl-3,5,9,11-tetraoxatricyclo [6.2.1.0-2,6-] undec-1-yl] -2 , 5,8,11-tetraoxatridecan-13-yl} -1H-1,2,3-triazol-4-yl) methoxy] -2-{[(1- {1-[(1S, 2R, 6R, 7R, 8S) -7- (acetylamino) -4,4-dimethyl-3,5,9,11-tetraoxatricyclo [6.2.1.0-2,6-] undec-1- Yl] -2,5,8,11-tetraoxatridecan-13-yl} -1H-1,2,3 To a solution of triazol-4-yl) methoxy] methyl} propan-2-yl) hexanamide (1-ag-1) (222 mg, 0.125 mmol) was added N, N-diisopropylethylamine (0.0873 mL, 0.501 mmol). ) And benzyl {6-[(2,5-dioxopyrrolidin-1-yl) oxy] -6-oxohexyl} carbamate
(Journal of Heterocyclic Chemistry, 23 (3), 901-3, 1986, 68.1 mg, 0.188 mmol) was added. The reaction was stirred at room temperature for 24 hours. After 24 hours, the reaction was concentrated under reduced pressure. The crude reaction mixture was purified using CombiFlash Rf (RediSep 12 g Gold silica gel column) eluting with a methanol / dichloromethane gradient from 0% to 20% to give the title compound as a gum. (250 mg, 99%). Method C: 3-minute operation LRMS [1 / 2M + 1 = 1010]. ¹ H-NMR (methanol-d ₄ ) δ: 7.98 (s, 3H), 7.22-7.40 (m, 5H), 5.22 (d, J = 1.2 Hz, 3H), 5 .06 (s, 2H), 4.51-4.61 (m, 12H), 4.29 (d, J = 5.9 Hz, 3H), 4.16 (t, J = 6.4 Hz, 3H) 3.86-3.96 (m, 12H), 3.83 (d, J = 7.8 Hz, 3H), 3.73-3.79 (m, 12H), 3.53-3.70 ( m, 36H), 3.04-3.19 (m, 4H), 2.17 (t, J = 7.4 Hz, 4H), 1.98 (s, 9H), 1.58 (td, J = 14.5, 7.6 Hz, 4H), 1.48 (s, 13H), 1.33 (s, 13H)

Benzyl [6-({6-[(1,3-bis [(1- {1-[(1S, 2R, 3R, 4R, 5S) -4- (acetylamino) -2,3-dihydroxy-6, 8-dioxabicyclo [3.2.1] oct-1-yl] -2,5,8,11-tetraoxatridecan-13-yl} -1H-1,2,3-triazol-4-yl ) Methoxy] -2-{[(1- {1-[(1S, 2R, 3R, 4R, 5S) -4- (acetylamino) -2,3-dihydroxy-6,8-dioxabicyclo [3. 2.1] oct-1-yl] -2,5,8,11-tetraoxatridecan-13-yl} -1H-1,2,3-triazol-4-yl) methoxy] methyl} propane-2 -Yl) amino] -6-oxohexyl} amino) -6-oxohexyl] carbame Doo (61)
Benzyl [6-({6-[(1,3-bis [(1- {1-[(1S, 2R, 6R, 7R, 8S) in acetic acid (8 mL), methanol (2 mL) and water (2 mL)]. ) -7- (acetylamino) -4,4-dimethyl-3,5,9,11-tetraoxatricyclo [6.2.1.0-2,6-] undec-1-yl] -2, 5,8,11-tetraoxatridecan-13-yl] -1H-1,2,3-triazol-4-yl) methoxy] 2-{[(1- {1- [1S, 2R, 6R, 7R , 8S) -7- (acetylamino) -4,4-dimethyl-3,5,9,11-tetraoxatricyclo [6.2.1.0-2,6-] undec-1-yl]- 2,5,8,11-tetraoxatridecan-13-yl] -1H-1,2,3-triazol-4-i } Methoxy] methyl} propan-2-yl) amino] -6-oxohexyl} amino) -6-oxohexyl] carbamate (1-ag-7) (250.0 mg, 0.124 mmol) was added for 36 hours 70 Heated to ° C. After 36 hours, the reaction was cooled to room temperature and concentrated under reduced pressure. The crude material was diluted with toluene and concentrated under reduced pressure. The crude material was diluted a second time with toluene and concentrated under reduced pressure to give the title compound as a gum (225 mg, 96%). Method C: 3-minute operation LRMS [1 / 2M + 1 = 950]. ¹ H-NMR (methanol-d ₄ ) δ: 7.98 (s, 3H), 7.22-7.41 (m, 5H), 5.21 (s, 3H), 5.05 (s, 2H) ), 4.57 (t, J = 5.0 Hz, 6H), 4.55 (s, 6H), 3.92-4.00 (m, 6H), 3.83-3.91 (m, 9H) ), 3.73-3.78 (m, 9H), 3.68-3.72 (m, J = 10.0, 4.1 Hz, 3H), 3.52-3.68 (m, 42H) , 3.05-3.17 (m, 4H), 2.16 (t, J = 7.3 Hz, 4H), 1.98 (s, 9H), 1.57-1.65 (m, 2H) , 1.53-1.57 (m, 2H), 1.43-1.52 (m, 4H), 1.24-1.39 (m, 4H)

6-amino-N- {6-[(1,3-bis [(1- {1-[(1S, 2R, 3R, 4R, 5S) -4- (acetylamino) -2,3-dihydroxy-6 , 8-dioxabicyclo [3.2.1] oct-1-yl] -2,5,8,11-tetraoxatridecan-13-yl} -1H-1,2,3-triazole-4- Yl) methoxy] -2-{[(1- {1-[(1S, 2R, 3R, 4R, 5S) -4- (acetylamino) -2,3-dihydroxy-6,8-dioxabicyclo [3 2.1] oct-1-yl] -2,5,8,11-tetraoxatridecan-13-yl] -1H-1,2,3-triazol-4-yl) methoxy] methyl} propane- 2-yl) amino-6-oxohexyl} hexaneamide acetate (62)
Benzyl [6-({6-[(1,3-bis [(1- {1-[(1S, 2R, 3R, 4R, 5S) -4- (acetylamino) -2,3-dihydroxy-6, 8-dioxabicyclo [3.2.1] oct-1-yl] -2,5,8,11-tetraoxatridecan-13-yl} -1H-1,2,3-triazol-4-yl ) Methoxy] -2-{[(1- {1-[(1S, 2R, 3R, 4R, 5S) -4- (acetylamino) -2,3-dihydroxy-6,8-dioxabicyclo [3. 2.1] oct-1-yl] -2,5,8,11-tetraoxatridecan-13-yl} -1H-1,2,3-triazol-4-yl) methoxy] methyl} propane-2 -Yl) amino] -6-oxohexyl} amino) -6-oxohexyl] carbame (61) (200 mg, 0.105 mmol) was dissolved in methanol (20 mL) and acetic acid (0.024 mL, 0.421 mmol), then the solution was dissolved in the following parameters (temperature = 50 ° C., flow rate = 1. 0 mL / min, pressure = total H ₂ (1 bar)) and was passed through an H cube using 10% palladium on carbon (small cartridge). The solution was collected and concentrated under reduced pressure to give the title compound as a gum (148 mg, 77%). Method C: LRMS operating for 3 minutes [M + 45 (formic acid) = 1809]. ¹ H-NMR (methanol-d ₄ ) δ: 8.02 (s, 3H), 5.23 (s, 3H), 4.59-4.63 (m, 6H), 4.58 (s, 6H) ), 3.97 (dd, J = 9.6, 5.3 Hz, 6H), 3.91 (d, J = 11.3, 4.7 Hz, 9H), 3.76-3.82 (m, 9H), 3.73 (dd, J = 10.1, 4.3 Hz, 3H), 3.56-3.70 (m, 42H), 3.16 (t, J = 6.8 Hz, 2H), 2.93 (t, J = 7.6 Hz, 2H), 2.16-2.29 (m, 4H), 2.01 (s, 9H), 1.92 (s, 3H), 1.62- 1.74 (m, 4H), 1.54-1.61 (m, 2H), 1.46-1.53 (m, 2H), 1.39-1.45 (m, 2H), 29-1. 8 (m, 2H)

[(1S, 2R, 6R, 7R, 8S) -7-azido-4,4-dimethyl-3,5,9,11-tetraoxatricyclo [6.2.1.0-2,6-] undeca -1-yl] methanol (1-d-1)
(1S, 2R, 3R, 4R, 5S) -4-azido-1- (hydroxymethyl) -6,8-dioxabicyclo [3.2.1] octane- in N, N-dimethylformamide (21 mL) To a solution of 2,3-diol (1) (2.52 g, 11.61 mmol) was added 2 2-dimethoxypropane (9.0 mL, 69.6 mmol), then (1S)-(+)-10-camphor. Sulfonic acid (1.08 g, 4.65 mmol) was added. The reaction was heated to 70 ° C. for 24 hours. After 24 hours, the reaction was cooled to room temperature before methanol (5 mL) was added followed by triethylamine (0.22 mL, 1.55 mmol) and the solution was stirred for 10 minutes before being concentrated under reduced pressure. The crude material was purified using CombiFlash Rf (RediSep 80g Gold silica gel column) eluting with a methanol / dichloromethane gradient from 0% to 20% to give the impure title compound. The crude material was purified using CombiFlash Rf (RediSep 40 g Gold silica gel column) eluting with a 0% to 100% ethyl acetate / heptane gradient to give the impure title compound ( 2419 mg, 81%). ¹ H-NMR (methanol-d ₄ ) δ: 5.42 (d, J = 1.6 Hz, 1H), 4.34-4.43 (m, 2H), 3.88-3.98 (m, 3H), 3.81-3.87 (m, 1H), 3.37 (dd, J = 6.2, 1.6 Hz, 1H), 1.54 (s, 3H), 1.42 (s, 3H)

(1S, 2R, 6R, 7R, 8S) -7-azido-4,4-dimethyl-1- (15-phenyl-2,5,8,11,14-pentaoxapentadec-1-yl) -3 , 5,9,11-tetraoxatricyclo [6.2.1.0-2,6-] undecane (1-d-2)
[(1S, 2R, 6R, 7R, 8S) -7-azido-4,4-dimethyl-3,5,9,11-tetraoxatricyclo [6.2.1.0] in tetrahydrofuran (5 mL). ~ 2,6- ~ undec-1-yl] methanol (1-d-1) (490 mg, 1.90 mmol) in 60% sodium hydroxide dispersion (127 mg, 3.2 mmol) in mineral oil at room temperature. Added at. The reaction was stirred under nitrogen for 30 minutes before 13-iodo-1-phenyl-2,5,8,11-tetraoxatridecane (1130 mg, 2.86 mmol) in tetrahydrofuran (2 mL) was added. The reaction was stirred overnight at room temperature. The next morning (18 hours), the reaction was quenched with water and extracted with ethyl acetate. The aqueous layer was washed twice more with ethyl acetate. The combined organic layers were washed with water, brine, dried over sodium sulfate, filtered and concentrated under reduced pressure. The crude material was purified using CombiFlash • Rf (ISCO • RediSep • gold • 40 g silica gel column) eluting with an ethyl acetate / heptane gradient from 0% to 100% to give the title compound as a gum Obtained as a product (336.0 mg, 34%).
Method C: LRMS running for 1.5 minutes [M + Na = 546]. ¹ H-NMR (methanol-d ₄ ) δ: 7.17-7.47 (m, 5H), 5.35 (d, J = 1.6 Hz, 1H), 4.55 (s, 2H), 4 .32-4.37 (m, 1H), 4.25-4.32 (m, 1H), 3.92 (d, J = 10.1 Hz, 1H), 3.88 (d, J = 8. 2Hz, 1H), 3.73-3.80 (m, 2H), 3.55-3.71 (m, 17H), 1.49 (s, 3H), 1.36 (s, 3H)

tert-Butyl [(1S, 2R, 6R, 7R, 8S) -1- (13-hydroxy-2,5,8,11-tetraoxatridec-1-yl) -4,4-dimethyl-3,5 , 9,11-Tetraoxatricyclo [6.2.1.0-2,6-] undec-7-yl] carbamate (1-ao-1)
In a 50 mL reactor, the starting material (1S, 2R, 6R, 7R, 8S) -7-azido-4,4-dimethyl-1- (15-phenyl-2,5,8,11,14-pentaoxa Pentadeca-1-yl) -3,5,9,11-tetraoxatricyclo [6.2.1.0-2,6-]] undecane (1-d-2) (310.0 mg, 0. 592 mmol) in methanol (6 mL) followed by di-tert-butyl-dicarbonate (162 mg, 0.74 mmol) and 10% palladium on carbon (50% wet weight / weight, 100.0 mg, 0.940 mmol). Added. The reactor was sealed and the reaction purged 3 times with nitrogen (50 psi) and then 2 times with hydrogen (50 psi), charged with hydrogen to 50 psi and stirred overnight. The next morning (24 hours), the reaction was filtered through a celite plug and washed with methanol. The filtrate was concentrated under reduced pressure. The crude material was purified using a CombiFlash Rf (RediSep 12 g silica gel column) eluting with a methanol / dichloromethane gradient from 0% to 20% to give the title compound as a gum (304 mg, 100 %). Method C: LRMS [M + Na = 530] running for 3 minutes. ¹ H-NMR (methanol-d ₄ ) δ: 5.22 (s, 1H), 4.28 (d, J = 5.9 Hz, 1H), 4.11 (t, J = 6.4 Hz, 1H) 3.93 (d, J = 10.1 Hz, 1H), 3.80-3.85 (m, 1H), 3.76 (d, J = 6.2 Hz, 1H), 3.74 (d, J = 3.9 Hz, 1H), 3.60-3.71 (m, 15H), 3.53-3.59 (m, 2H), 1.50 (s, 3H), 1.45 (s, 9H), 1.34 (s, 3H)

1-{(1S.2R.6R.7R.8S) -7-[(tert-butoxycarbonyl) amino] -4,4-dimethyl-3,5,9,11-tetraoxatricyclo [6.2. 1.0 to 2,6 to] undec-1-yl} -2,5,8,11-tetraoxatridecan-13-ylmethanesulfonate (1-ap-1)
Tert-Butyl [(1S, 2R, 6R, 7R, 8S) -1- (13-hydroxy-2,5,8,11-tetraoxatridec-1-yl) -4 in didichloromethane (2 mL) , 4-Dimethyl-3,5,9,11-tetraoxatricyclo [6.2.1.0-2,6-]-undec-7-yl] carbamate (1-ao-1) (300.0 mg, To a solution of 0.591 mmol) was added triethylamine (0.332 ml, 2.36 mmol), cooled to 0 ° C. using an ice bath, followed by methanesulfonyl chloride (0.055 mL, 0.71 mmol). The reaction was gradually warmed to room temperature and stirred at room temperature for 20 hours. After 20 hours, the reaction was quenched with water and extracted. The layers were separated and the aqueous layer was further extracted with dichloromethane. The combined organic layers were washed with brine, dried over magnesium sulfate, filtered and concentrated under reduced pressure to give the title compound as an oil (339 mg, 98%). Method C: Run for 3 minutes LRMS [M + Na = 530] ¹ H-NMR (methanol-d ₄ ) δ: 5.22 (s, 1H), 4.33-4.43 (m, 2H), 4.28 ( d, J = 5.9 Hz, 1H), 4.11 (t, J = 6.4 Hz, 1H), 3.93 (d, J = 10.1 Hz, 1H), 3.80-3.86 (m) , 1H), 3.72-3.79 (m, 4H), 3.61-3.70 (m, 12H), 3.58 (d, J = 5.9 Hz, 1H), 3.11 (s) , 3H), 1.50 (s, 3H), 1.45 (s, 9H), 1.34 (s, 3H)

tert-Butyl [(1S, 2R, 6R, 7R, 8S) -1- (13-azido-2,5,8,11-tetraoxatridec-1-yl) -4,4-dimethyl-3,5 , 9,11-tetraoxatricyclo [6.2.1.0-2,6-] undec-7-yl] carbamate (1-aq-1)
1-{(1S, 2R, 6R, 7R, 8S) -7-[(tert-butoxycarbonyl) amino] -4,4-dimethyl-3,5 in N, N-dimethylformamide (1.5 mL). , 9,11-tetraoxatricyclo [6.2.1.0-2,6-] undec-1-yl} -2,5,8,11-tetraoxatridecan-13-ylmethanesulfonate (1 To a solution of -ap-1) (339 mg 0.579 mmol) was added sodium azide (67.7 mg, 1.04 mmol) and the reaction was heated to 100 ° C. overnight in a sealed 5 mL microwave vial. After 18 hours, the reaction was cooled to room temperature and the reaction was diluted with water and extracted three times with ethyl acetate. The combined organic layers were washed with water, brine, dried over sodium sulfate, filtered and concentrated under reduced pressure. The crude material was purified using CombiFlash® Rf (RediSep 12g Gold silica gel column) eluting with a methanol / dichloromethane gradient from 0% to 20% to give the title compound as a gum. (246 mg, 80%). ¹ H-NMR (methanol-d ₄ ) δ: 5.24 (s, 1H), 4.30 (d, J = 5.9 Hz, 1H), 4.13 (t, J = 6.4 Hz, 1H) 3.95 (d, J = 9.8 Hz, 1H), 3.82-3.88 (m, 1H), 3.75-3.80 (m, 2H), 3.53-3.74 ( m, 15H), 3.39 (t, J = 4.9 Hz, 2H), 1.52 (s, 3H), 1.47 (s, 9H), 1.36 (s, 3H)

tert-butyl {(1S, 2R, 6R, 7R, 8S) -1- [13- (4-{[2-{[4- (benzyloxy) butanoyl] amino} -3-{[1- (1- {(1S, 2R, 6R, 7R, 8S) -7-[(tert-butoxycarbonyl) amino] -4,4-dimethyl-3,5,9,11-tetraoxytricyclo [6.2.1. 0-2,6- ~ undec-1-yl} -2,5,8,11-tetraoxytridecan-13-yl) -1H-1,2,3-triazol-4-yl] methoxy} -2 -({[1- (1-{(1S, 2R, 6R, 7R, 8S) -7-[(tert-butoxycarbonyl) amino] -4,4-dimethyl-3,5,9,11-tetraoxa Tricyclo [6.2.1.0-2,6-] undec-1-yl} -2,5, , 11-tetraoxatridecan-13-yl) -1H-1,2,3-triazol-4-yl] methoxy} methyl) propoxy] methyl} -1H-1,2,3-triazol-1-yl) -2,5,8,11-tetraoxatridec-1-yl] -4,4-dimethyl-3,5,9,11-tetraoxatricyclo [6.2.1.0-2,6- ] Undec-7-yl} carbamate (1-ar-1)
To a 20 mL vial with a stir bar, add 4- (benzyloxy) -N- {1,3-bis (prop-2-yn-1-yloxy) -2-[(prop-2-yn-1-yloxy). ) Methyl) propan-2-yl} butanamide (1-v-1) (45.0 mg, 0.11 mmol) was charged to t-butanol (3 mL) and water (1.5 mL, deionized water). Of tert-butyl [(1S, 2R, 6R, 7R, 8S) -1- (13-azido-2,5,8,11-tetraoxatridec-1-yl) -4,4-dimethyl-3 , 5,9,11-tetraoxatricyclo [6.2.1.0-2,6-] undec-7-yl] carbamate (1-aq-1) (192 mg, 0.361 mmol) was added. After purging the reaction with nitrogen for 5 minutes, sodium ascorbate (66.3 mg, 0.328 mmol) was added and a solution of copper (II) sulfate (5.24 mg, deionized water) in water (5.24 mg, 0.0328 mmol) was added dropwise. The reaction was stirred at room temperature for 20 hours. After 20 hours, the reaction was cooled to room temperature and the reaction mixture was added to saturated ammonium chloride (30 mL) and concentrated ammonium hydroxide (2 mL) to quench the reaction and extracted three times with dichloromethane (15 mL). The combined organic layers were dried over sodium sulfate, filtered and concentrated under reduced pressure. The crude material was purified using CombiFlash Rf (RediSep 12 g Gold silica gel column) eluting with a methanol / dichloromethane gradient from 0% to 20% to give the title compound as a gum. (165 mg, none, 75%). Method C: 3-minute operation LRMS [1 / 2M + 1 = 1005. ¹ H-NMR (methanol-d ₄ ) δ: 7.97 (s, 3H), 7.17-7.43 (m, 5H), 5.21 (s, 3H), 4.52-4.60 (M, 12H), 4.45 (s, 2H), 4.25 (d, J = 5.9 Hz, 3H), 4.10 (t, J = 6.2 Hz, 3H), 3.85-3 .93 (m, 9H), 3.71-3.82 (m, 15H), 3.63-3.69 (m, 6H), 3.53-3.62 (m, 33H), 3.48 (T, J = 6.2 Hz, 2H), 2.27 (t, J = 7.2 Hz, 2H), 1.74-1.96 (m, 2H), 1.49 (s, 9H), 1 .45 (s, 27H), 1.32 (s, 9H)

4- (benzyloxy) -N- (1,3-bis [(1- {1-[(1S, 2R, 3R,
4R, 5S) -4-amino-2,3-dihydroxy-6,8-dioxabicyclo [3.2.1] oct-1-yl] -2,5,8,11-tetraoxatridecane-13 -Yl} -1H-1,2,3-triazol-4-yl) methoxy] -2-{[(1- {1-[(1S, 2R, 3R, 4R, 5S) -4-amino-2, 3-dihydroxy-6,8-dioxabicyclo [3.2.1] oct-1-yl] -2,5,8,11-tetraoxatridecan-13-yl} -1H-1,2,3 -Triazol-4-yl) methoxy] methyl} propan-2-yl) butanamide (63)
Tert-Butyl {(1S, 2R, 6R, 7R, 8S) -1- [13- (4-{[2- {] in acetic acid (5 mL), methanol (1.5 mL) and water (1.5 mL). [4- (Benzyloxy) butanoyl] amino} -3-{[1- (1-{(1S, 2R, 6R, 7R, 8S) -7-[(tert-butoxycarbonyl) amino] -4,4- Dimethyl-3,5,9,11-tetraoxytricyclo [6.2.1.0-2,6-]-undec-1-yl} -2,5,8,11-tetraoxytridecan-13 Yl) -1H-1,2,3-triazol-4-yl] methoxy} -2-({[1- (1-{(1S, 2R, 6R, 7R, 8S) -7-[(tert-butoxy) Carbonyl) amino] -4,4-dimethyl-3,5,9,11-tetraoxato Cyclo [6.2.1.0-2,6-] undec-1-yl} -2,5,8,11-tetraoxatridecan-13-yl) -1H-1,2,3-triazole- 4-yl] methoxy} methyl) propoxy] methyl} -1H-1,2,3-triazol-1-yl) -2,5,8,11-tetraoxatridec-1-yl] -4,4- Dimethyl-3,5,9,11-tetraoxatricyclo [6.2.1.0-2,6-]-undec-7-yl} carbamate (1-ar-1) (150.0 mg, 0.0747 mmol ) Was heated to 70 ° C. for 18 hours. After 18 hours, the reaction was cooled to room temperature and concentrated under reduced pressure. The crude material was diluted with dichloromethane (10 mL) and methanol (4 mL) and to this was added 4.0 M hydrogen chloride in dioxane (2.0 mL, 8 mmol). The reaction mixture was stirred at room temperature overnight. After 18 hours, the reaction was concentrated under reduced pressure. The crude material was diluted with ethyl acetate (1 mL), to which heptane (10 mL) was added and concentrated under reduced pressure. This material was then placed under high vacuum for 18 hours to give the title compound (139.0 mg, 103%). Method C: 3-minute operation LRMS [1 / 2M + 1 = 795]. ¹ H-NMR (methanol-d ₄ ) δ: 8.09 (s, 3H), 7.27-7.39 (m, 5H), 5.48 (s, 3H), 4.57-4.66 (M, 12H), 4.47 (s, 2H), 3.98 (d, J = 9.8 Hz, 3H), 3.90-3.95 (m, 9H), 3.82-3.89 (M, 6H), 3.79 (s, 6H), 3.76 (d, J = 8.2 Hz, 3H), 3.71 (d, J = 9.8 Hz, 3H), 3.57-3 .69 (m, 36H), 3.50 (t, J = 6.2 Hz, 2H), 3.21 (d, J = 9.4 Hz, 3H), 2.29 (t, J = 7.2 Hz, 2H), 1.85 (quin, J = 6.8 Hz, 2H)

(1S, 2R, 3R, 4R, 5S) -4- (acetylamino) -1- (13- {4-[(3-[(1- {1-[(1S, 2R, 3R, 4R, 5S) -4- (acetylamino) -2,3-bis (acetyloxy) -6,8-dioxabicyclo [3.2.1] oct-1-yl] -2,5,8,11-tetraoxatri Decan-13-yl} -1H-1,2,3-triazol-4-yl) methoxy] -2-{[(1- {1-[(1S, 2R, 3R, 4R, 5S) -4- ( Acetylamino) -2,3-bis (acetyloxy) -6,8-dioxabicyclo [3.2.1] oct-1-yl] -2,5,8,11-tetraoxatridecane-13 Yl} -1H-1,2,3-triazol-4-yl) methoxy] methyl} -2-{[4- (benzyl) Oxy) butanoyl] amino} propoxy) methyl] -1H-1,2,3-triazol-1-yl} -2,5,8,11-tetraoxatridec-1-yl) -3- (acetyloxy) -6,8-dioxabicyclo [3.2.1] oct-2-yl acetate (1-as-1)
4- (Benzyloxy) -N- (1,3-bis [(1- {1-[(1S, 2R, 3R, 4R, 5S) -4-amino-2,3-dihydroxy-6,8-di Oxabicyclo [3.2.1] oct-1-yl] -2,5,8,11-tetraoxatridecan-13-yl} -1H-1,2,3-triazol-4-yl) methoxy] -2-{[(1- {1-[(1S, 2R, 3R, 4R, 5S) -4-amino-2,3-dihydroxy-6,8-dioxabicyclo [3.2.1] octa- 1-yl] -2,5,8,11-tetraoxatridecan-13-yl} -1H-1,2,3-triazol-4-yl) methoxy] methyl} propan-2-yl) butanamide (63 ) (80 mg, 0.044 mmol) was added to pyridine (anhydrous) (1.5 mL, 1 It was dissolved in mmol), which was added acetic anhydride (0.125 mL, 1.33 mmol) at room temperature. The reaction was then heated at 50 ° C. overnight. The next morning, the reaction was concentrated under reduced pressure. The crude material was purified using CombiFlash Rf (RediSep 12 g Gold silica gel column) eluting with a methanol / dichloromethane gradient from 0% to 20% to give the crude title compound. The crude title compound was purified using CombiFlash Rf (RediSep 4g Gold silica gel column) eluting with a methanol / dichloromethane gradient from 0% to 20% to give the title compound as a gum. (54.0 mg, 62%). Method C: LRMS [1 / 2M + 1 = 984] running for 1.5 minutes. ¹ H-NMR (methanol-d ₄ ) δ: 7.97 (s, 3H), 7.19-7.42 (m, 5H), 5.44 (d, J = 4.3 Hz, 3H), 5 .31 (s, 3H), 5.10 (dd, J = 10.3, 4.1 Hz, 3H), 4.51-4.64 (m, 12H), 4.45 (s, 2H), 4 .18 (d, J = 10.1 Hz, 3H), 3.99 (d, J = 8.6 Hz, 3H), 3.88 (t, J = 4.9 Hz, 6H), 3.68-3. 82 (m, 12H), 3.52-3.64 (m, 39H), 3.48 (t, J = 6.2 Hz, 2H), 2.26 (t, J = 7.4 Hz, 2H), 2.15 (s, 9H), 1.94 (d, J = 1.2 Hz, 18H), 1.84 (t, J = 6.8 Hz, 2H).

(1S, 2R, 3R, 4R, 5S) -4- (acetylamino) -1- {13- [4-({3-[(1- {1-[(1S, 2R, 3R, 4R, 5S) -4- (acetylamino) -2,3-bis (acetyloxy) -6,8-dioxabicyclo [3.2.1] oct-1-yl] -2,5,8,11-tetraoxatri Decan-13-yl} -1H-1,2,3-triazol-4-yl) methoxy] -2-{[(1- {1-[(1S, 2R, 3R, 4R, 5S) -4- ( Acetylamino) -2,3-bis (acetyloxy) -6,8-dioxabicyclo [3.2.1] oct-1-yl] -2,5,8,11-tetraoxatridecane-13 Yl} -1H-1,2,3-triazol-4-yl) methoxy] methyl} -2-[(4-hydroxy Butanoyl) amino] propoxy} methyl) -1H-1,2,3-triazol-1-yl] -2,5,8,11-tetraoxatridec-1-yl} -3- (acetyloxy) -6 , 8-dioxabicyclo [3.2.1] oct-2-yl acetate (1-at-1)
(1S, 2R, 3R, 4R, 5S) -4- (acetylamino) -1- (13- {4-[(3-[(1- {1-[(1S, 2R, 3R, 4R, 5S) -4- (acetylamino) -2,3-bis (acetyloxy) -6,8-dioxabicyclo [3.2.1] oct-1-yl] -2,5,8,11-tetraoxatri Decan-13-yl} -1H-1,2,3-triazol-4-yl) methoxy] -2-{[(1- {1-[(1S, 2R, 3R, 4R, 5S) -4- ( Acetylamino) -2,3-bis (acetyloxy) -6,8-dioxabicyclo [3.2.1] oct-1-yl] -2,5,8,11-tetraoxatridecane-13 Yl} -1H-1,2,3-triazol-4-yl) methoxy] methyl} -2-{[4- (benzyl) Oxy) butanoyl] amino} propoxy) methyl] -1H-1,2,3-triazol-1-yl} -2,5,8,11-tetraoxatridec-1-yl) -3- (acetyloxy) -6,8-dioxabicyclo [3.2.1] oct-2-yl acetate (1-as-1) (54.0 mg, 0.027 mmol) was dissolved in methanol (10.0 mL) and then The solution is put into an H-cube using 10% palladium on carbon (small cartridge) using the following parameters (temperature = 60 ° C., flow rate = 1.0 mL / min, pressure = total H ₂ (1 atm)). I passed. The solution was collected and concentrated under reduced pressure to give the title compound as a gum (51.0 mg, 99%). Method C: LRMS [M + Na = 1899] running for 3 minutes. ¹ H-NMR (methanol-d ₄ ) δ: 7.98 (s, 3H), 5.44 (d, J = 4.3 Hz, 3H), 5.32 (s, 3H), 5.10 (dd , J = 10.5, 3.9 Hz, 3H), 4.41-4.66 (m, 12H), 4.18 (d, J = 10.5 Hz, 3H), 3.99 (d, J = 8.6 Hz, 3H), 3.90 (t, J = 5.1 Hz, 6H), 3.68-3.83 (m, 12H), 3.51-3.67 (m, 41H), 2. 24 (t, J = 7.6 Hz, 2H), 2.15 (s, 9H), 1.94 (s, 18H), 1.69-1.83 (m, 2H)

N- (1,3-bis [(1- {1-[(1S, 2R, 3R, 4R, 5S) -4- (acetylamino) -2,3-dihydroxy-6,8-dioxabicyclo [3 2.1] oct-1-yl] -2,5,8,11-tetraoxatridecan-13-yl} -1H-1,2,3-triazol-4-yl) methoxy] -2- { [(1- {1-[(1S, 2R, 3R, 4R, 5S) -4- (acetylamino) -2,3-dihydroxy-6,8-dioxabicyclo [3.2.1] octa-1 -Yl] -2,5,8,11-tetraoxatridecan-13-yl} -1H-1,2,3-triazol-4-yl) methoxy] methyl} propan-2-yl) -4-hydroxy Butanamide (64)
(1S, 2R, 3R, 4R, 5S) -4- (Acetylamino) -1- {13- [4-({3-[(1- {) in methanol (0.154 mL mg, 0.0770 mmol) 1-[(1S, 2R, 3R, 4R, 5S) -4- (acetylamino) -2,3-bis (acetyloxy) -6,8-dioxabicyclo [3.2.1] oct-1- Yl] -2,5,8,11-tetraoxatridecan-13-yl} -1H-1,2,3-triazol-4-yl) methoxy] -2-{[(1- {1-[( 1S, 2R, 3R, 4R, 5S) -4- (acetylamino) -2,3-bis (acetyloxy) -6,8-dioxabicyclo [3.2.1] oct-1-yl] -2 , 5,8,11-tetraoxatridecan-13-yl} -1H-1,2,3-tri Azol-4-yl) methoxy] methyl} -2-[(4-hydroxybutanoyl) amino] propoxy} methyl) -1H-1,2,3-triazol-1-yl] -2,5,8,11 -Tetraoxatridec-1-yl} -3- (acetyloxy) -6,8-dioxabicyclo [3.2.1] oct-2-yl acetate (1-at-1) (8.5 mg, To a solution of 0.0045 mmol) 0.5 M sodium methoxide (0.154 mL, 0.0770 mmol) in methanol (1 mL) was added and the reaction was stirred at room temperature for 3 hours. After 3 hours, the reaction was neutralized by adding Amberlyst 15 ion exchange resin (CAS # = 39389-20-3, RS-106008) rinsed 3 times with methanol until pH = 5. The reaction mixture was filtered and the resin was rinsed twice with methanol. The filtrate was concentrated under reduced pressure to give the title compound as a gum (1.5 mg, 20%). Method C: LRMS run for 3 minutes [M + 45 (formic acid) = 1668]. ¹ H-NMR (methanol-d ₄ ) δ: 8.00 (s, 3H), 5.21 (s,
3H), 4.53-4.65 (m, 12H), 3.92-4.00 (m, 6H), 3.89 (dd, J = 11.1, 4.9 Hz, 9H), 74-3.79 (m,
9H), 3.71 (dd, J = 9.8, 4.3 Hz, 3H), 3.52-3.69 (m, 44H), 2.24 (t, J = 7.4 Hz, 2H), 1.99 (s, 9H), 1.76 (quin, J = 6.9 Hz, 2H).

The orthoester linker illustrated by compound (66) of Scheme 4b and described generally in Scheme 3b is (I-aw-1) (H. Bruyere et al.) To produce (I-ax-1). al, Bioorg. Med. Chem. Lett., 20, 2200-2203, (2010)), in a suitable alcohol such as (I-an-1), and in a suitable solvent such as toluene, p- One skilled in the art would be able to synthesize by utilizing a suitable acid, such as pyridinium toluenesulfonate, under reflux conditions. Deprotection of (I-ax-1) can be achieved under basic conditions known to those skilled in the art (such as catalytic potassium carbonate in methanol) which will yield compound (65) . Further functionalization of (65) can be achieved to produce the additional compounds claimed in the present invention. Thus, using an appropriate base such as N, N-diisopropylethylamine, an appropriate acid and coupling agent (known to those skilled in the art) or an activated ester such as (Is-1) The compound (66) can be produced by treating (65) with a solvent such as N, N-dimethylformamide (for example, hydroxysuccinimide).

In a similar manner, as shown in Scheme 5b, one of ordinary skill in the art can use appropriate reaction alcohols such as (I-an-1) and (I-av) by using the reaction conditions described above for Scheme 4b. The compound (66) could be synthesized using -1).

N- (1,3-dihydroxypropan-2-yl) -6- (pyridin-2-yldisulfanyl) hexanamide (1-au-1)
1-{[6- (Pyridin-2-yldisulfanyl) hexanoyl] oxy} pyrrolidine-2,5-dione (1-s-1) (518 mg, 2.01 mmol) in N, N-dimethylformamide (7 mL) To this solution, N- (3-dimethylaminopropyl) -N′-ethylcarbodiimide hydrochloride (463 mg, 2.42 mmol) and 1-hydroxybenzotriazole (326 mg, 2.42 mmol) were added and stirred at room temperature for 1 hour. . After 1 hour, 2-aminopropane-1,3-diol (183 mg, 2.01 mmol) was added, followed by N, N-diisopropylethylamine (1.05 mL, 6.04 mmol). The reaction was stirred overnight at room temperature. After 18 hours, the reaction was diluted with water and extracted three times with dichloromethane. The combined organic layers were washed with brine, dried over magnesium sulfate, filtered and concentrated under reduced pressure. The crude material was eluted using a CombiFlash Rf (RediSep 24 g silica gel column) with a methanol / dichloromethane gradient from 0% to 20% to give the title compound (368 mg, 55%). Method C: LRMS running for 1.5 minutes [M + 45 (formic acid) = 375]. ¹ H-NMR (methanol-d ₄ ) δ: 8.38 (d, J = 4.3 Hz, 1H), 7.84-7.89 (m, 1H), 7.77-7.83 (m, 1H), 7.21 (t, J = 5.7 Hz, 1H), 3.92 (quin, J = 5.5 Hz, 1H), 3.51-3.70 (m, 4H), 2.82 ( t, J = 7.2 Hz, 2H), 2.21 (t, J = 7.4 Hz, 2H), 1.71 (quin, J = 7.3 Hz, 2H), 1.60 (quin, J = 7 .5Hz, 2H), 1.37-1.51 (m, 2H)

N- (2-methoxy-1,3-dioxane-5-yl) -6- (pyridin-2-yldisulfanyl) hexanamide (1-av-1)
N- (1,3-dihydroxypropan-2-yl) -6- (pyridin-2-yldisulfanyl) hexanamide (1- To a mixture of au-1) (280 mg, 0.847 mmol), 7-toluenesulfonic acid monohydrate (1.78 mg, 0.00847 mmol) was added. The reaction was stirred at room temperature for 3 hours. After 3 hours, TLC showed almost complete consumption of starting material. The reaction is diluted with dichloromethane, washed with saturated aqueous sodium bicarbonate (3 × 1 mL), brine (1 mL), dried over anhydrous potassium carbonate, filtered and concentrated under reduced pressure to give the crude title compound. Obtained (263.0 mg, 83.3%). Method C: Run for 3 minutes (basic mode: column: base: Waters Acquity UPLC BEH, 2.1 mm × 50 mm, C18, 1.8 μm. Mobile phase A: 0.1% ammonia in water (v / v). B: 0.1% ammonia / acetonitrile (v / v)). LRMS [M + 45 = 417]. A 1: 1 mixture of cis / trans isomers.
Isomer 1: ¹ H-NMR (methanol-d ₄ ) δ: 8.41 (d, J = 4.7 Hz, 1H), 7.86-7.92 (m, 1H), 7.80-7. 86 (m, 1H), 7.20-7.27 (m, 1H), 5.31 (s, 1H), 4.29 (d, J = 2.7 Hz, 1H), 3.92-3. 96 (m, 1H), 3.83 (br.s, 1H), 3.63 (d, J = 5.1 Hz, 1H), 3.59 (dd, J = 11.5, 3.7 Hz, 1H ), 3.42 (s, 3H), 2.85 (t, J = 7.2 Hz, 2H), 2.21-2.30 (m, 2H), 1.68-1.80 (m, 2H) ), 1.56-1.67 (m, 2H), 1.39-1.53 (m, 2H)
Isomer 2: ¹ H-NMR (methanol-d ₄ ) δ: 8.41 (d, J = 4.7 Hz, 1H), 7.86-7.92 (m, 1H), 7.80-7. 86 (m, 1H), 7.20-7.27 (m, 1H), 5.27 (s, 1H), 4.26 (d, J = 2.7 Hz, 1H), 3.92-3. 96 (m, 2H), 3.86-3.91 (m, 1H), 3.59 (dd, J = 11.5, 3.7 Hz, 1H), 3.38 (s, 3H), 85 (t, J = 7.2 Hz, 2H), 2.21-2.30 (m, 2H), 1.68-1.80 (m, 2H), 1.56-1.67 (m, 2H) ), 1.39-1.53 (m, 2H).

(1S, 2R, 3R, 4R, 5S) -4- (acetylamino) -2- (acetyloxy) -1- {13- [4- (12,12-bis {[(1- {1-[( 1S, 2R, 3R, 4R, 5S) -4- (acetylamino) -2,3-bis (acetyloxy) -6,8-dioxabicyclo [3.2.1] oct-1-yl] -2 , 5,8,11-tetraoxatridecan-13-yl} -1H-1,2,3-triazol-4-yl) methoxy] methyl} -3,10-dioxo-1-phenyl-2,14- Dioxa-4,11-diazapentadecan-15-yl) -1H-1,2,3-triazol-1-yl] -2,5,8,11-tetraoxatridec-1-yl} -6, 8-dioxabicyclo [3.2.1] oct-3-yl acetate ( -az-1)
Benzyl {6-[(1,3-bis [(1- {1-[(1S, 2R, 6R, 7R, 8S) -7- (acetylamino) -4,4-dimethyl-3,5,9, 11-tetraoxatricyclo [6.2.1.0-2,6-] undec-1-yl] -2,5,8,11-tetraoxatridecan-13-yl} -1H-1,2 , 3-Triazol-4-yl) methoxy] -2-{[(1- {1-[(1S, 2R, 3R, 4R, 5S) -4- (acetylamino) -2,3-dihydroxy-6, 8-dioxabicyclo [3.2.1] oct-1-yl] 2,5,8,11-tetraoxatridecan-13-yl} -1H-1,2,3-triazol-4-yl) Methoxy] methyl} propan-2-yl) amino] -6-oxohexyl} carbamate (47) 1690.0Mg, was dissolved 0.9463Mmol) in anhydrous pyridine (20mL, 250mmol), which acetic anhydride (2.68 mL, 28.4 mmol) was added at room temperature. The reaction was then heated to 50 ° C. overnight. The next morning, the reaction was concentrated under reduced pressure. The crude material was purified using CombiFlash Rf (RediSep 40 g Gold silica gel column) eluting with a methanol / dichloromethane gradient from 0% to 20% to give the title compound as a gum. (1172 mg, 62.8%).
Method C: 3-minute operation LRMS [1 / 2M + 1 = 1020]. ¹ H-NMR (methanol-d ₄ ) δ: 7.97 (s, 3H), 7.22-7.40 (m, 5H), 5.44 (d, J = 3.9 Hz, 3H), 5 .32 (s, 3H), 5.10 (dd, J = 10.5, 4.3 Hz, 3H), 5.05 (s, 2H), 4.56-4.60 (m, 6H), 4 .55 (s, 6H), 4.18 (d, J = 10.5 Hz, 3H), 3.99 (d, J = 8.6 Hz, 3H), 3.89 (t, J = 5.1 Hz, 6H), 3.71-3.80 (m, 12H), 3.51-3.65 (m, 39H), 3.09 (q, J = 6.2 Hz, 2H), 2.16-2. 19 (m, 2H), 2.15 (s, 9H), 1.94 (d, J = 1.6 Hz, 18H), 1.52-1.61 (m, 2H), 1.42-1. 2 (m, 2H), 1.33 (d, J = 7.0Hz, 2H)

(1S, 2R, 3R, 4R, 5S) -4- (acetylamino) -1- {13- [4-({3-[(1- {1-[(1S, 2R, 3R, 4R, 5S) -4- (acetylamino) -2,3-bis (acetyloxy) -6,8-dioxabicyclo [3.2.1] oct-1-yl] -2,5,8,11-tetraoxatri Decan-13-yl} -1H-1,2,3-triazol-4-yl) methoxy] -2-{[(1- {1-[(1S, 2R, 3R, 4R, 5S) -4- ( Acetylamino) -2,3-bis (acetyloxy) -6,8-dioxabicyclo [3.2.1] oct-1-yl] -2,5,8,11-tetraoxatridecane-13 Yl} -1H-1,2,3-triazol-4-yl) methoxy] methyl} -2-[(6-aminohe Sanoyl) amino] propoxy} methyl) -1H-1,2,3-triazol-1-yl] -2,5,8,11-tetraoxatridec-1-yl} -3- (acetyloxy) -6 , 8-dioxabicyclo [3.2.1] oct-2-yl acetate (I-ba-1)
(1S, 2R, 3R, 4R, 5S) -4- (acetylamino) -2- (acetyloxy) -1- {13- [4- (12,12-bis {[( 1- {1-[(1S, 2R, 3R, 4R, 5S) -4- (acetylamino) -2,3-bis (acetyloxy) -6,8-dioxabicyclo [3.2.1] octa -1-yl] -2,5,8,11-tetraoxatridecan-13-yl} -1H-1,2,3-triazol-4-yl) methoxy] methyl} -3,10-dioxo-1 -Phenyl-2,14-dioxa-4,11-diazapentadecan-15-yl) -1H-1,2,3-triazol-1-yl] -2,5,8,11-tetraoxatrideca- 1-yl} -6,8-dioxabicyclo [3.2.1] oct 3-yl acetate (I-az-1) ( 930.0mg, 0.456mmol) was treated with the following parameters (temperature = 50 ° C., flow rate = 1.0 mL / min, pressure = total H ₂ (1 bar) ) Was passed through an H-cube using 10% palladium on carbon (small cartridge). The solution was collected and concentrated under reduced pressure to give the title compound as a gum (837 mg, 96%). Method C: LRMS operating for 3 minutes [M + 45 (formic acid) -1 = 1948]. ¹ H-NMR (methanol-d ₄ ) δ: 7.9 (s, 3H), 5.44 (d, J = 4.3 Hz, 3H), 5.32 (s, 3H), 5.10 (dd , J = 10.5, 4.3 Hz, 3H), 4.59 (t, J = 5.1 Hz, 6H), 4.56 (s, 6H), 4.18 (d, J = 10.1 Hz, 3H), 3.99 (d, J = 8.2 Hz, 3H), 3.90 (t, J = 5.1 Hz, 6H), 3.71-3.82 (m, 12H), 3.53- 3.66 (m, 39H), 2.76 (t, J = 7.4 Hz, 2H), 2.16-2.23 (m, 2H), 2.15 (s, 9H), 1.94 ( s, 18H), 1.48-1.64 (m, 4H), 1.29-1.43 (m, 2H)

Ethyl 7-({6-[(1,3-bis [(1- {1-[(1S, 2R, 3R, 4R, 5S) -4- (acetylamino) -2,3-bis (acetyloxy ) -6,8-dioxabicyclo [3.2.1] oct-1-yl] -2,5,8,11-tetraoxatridecan-13-yl} -1H-1,2,3-triazole -4-yl) methoxy] -2-{[(1- {1-[(1S, 2R, 3R, 4R, 5S) -4- (acetylamino) -2,3-bis (acetyloxy) -6, 8-dioxabicyclo [3.2.1] oct-1-yl] -2,5,8,11-tetraoxatridecan-13-yl} -1H-1,2,3-triazol-4-yl ) Methoxy] methyl} propan-2-yl) amino] -6-oxohexyl}} amino) -7-oxy Heptanoate (I-bb-1)
(1S, 2R, 3R, 4R, 5S) -4- (acetylamino) -1- {13- [4-({3-[() in N, N-dimethylformamide (2 mL) and tetrahydrofuran (2 mL). 1- {1-[(1S, 2R, 3R, 4R, 5S) -4- (acetylamino) -2,3-bis (acetyloxy) -6,8-dioxabicyclo [3.2.1] octa -1-yl] -2,5,8,11-tetraoxatridecan-13-yl} -1H-1,2,3-triazol-4-yl) methoxy] -2-{[(1- {1 -[(1S, 2R, 3R, 4R, 5S) -4- (acetylamino) -2,3-bis (acetyloxy) -6,8-dioxabicyclo [3.2.1] oct-1-yl ] -2,5,8,11-tetraoxatridecan-13-yl} -1H- , 2,3-triazol-4-yl) methoxy] methyl} -2-[(6-aminohexanoyl) amino] propoxy} methyl) -1H-1,2,3-triazol-1-yl] -2, 5,8,11-Tetraoxatridec-1-yl} -3- (acetyloxy) -6,8-dioxabicyclo [3.2.1] oct-2-yl acetate (1-ba-1) To a solution of (200.0 mg, 0.105 mmol), ethyl 7-[(2,5-difluorophenyl) dioxopyrrolidin-1-yl) oxy] -7-oxoheptanoate
(40.3 mg, 0.126 mmol) and N, N-diisopropylethylamine (0.0732 mL, 0.420 mmol) were added and the reaction was stirred at room temperature for 24 hours. After 24 hours, the reaction was concentrated under reduced pressure. The crude material was purified using CombiFlash® Rf (RediSep 12g Gold silica gel column) eluting with a methanol / dichloromethane gradient from 0% to 20% to give the title compound as a gum. (140.0 mg, 64.3%). Method C: 3-minute operation LRMS [1 / 2M + 1 = 1038]. ¹ H-NMR (methanol-d ₄ ) δ: 7.98 (s, 3H), 5.44 (d, J = 4.3 Hz, 3H), 5.32 (s, 3H), 5.10 (dd , J = 10.5, 4.3 Hz, 3H), 4.57-4.61 (m, 6H), 4.56 (s, 6H), 4.18 (d, J = 9.8 Hz, 3H) 4.11 (q, J = 7.2 Hz, 2H), 3.99 (d, J = 8.6 Hz, 3H), 3.90 (t, J = 4.9 Hz, 6H), 3.71- 3.83 (m, 12H), 3.53-3.65 (m, 39H), 3.08-3.17 (m, 2H), 2.31 (t, J = 7.4 Hz, 2H), 2.16-2.22 (m, 4H), 2.15 (s, 9H), 1.94 (d, J = 1.6 Hz, 18H), 1.53-1.66 (m, 6H), 1.43-1.52 (m, 2H), 1.28-1.40 (m, 4H), 1.24 (t, J = 7.2 Hz, 3H)

7-({6-[(1,3-bis [(1- {1-[(1S, 2R, 3R, 4R, 5S) -4- (acetylamino) -2,3-dihydroxy-6,8- Dioxabicyclo [3.2.1] oct-1-yl] -2,5,8,11-tetraoxatridecan-13-yl} -1H-1,2,3-triazole-4-yl) methoxy ] -2-{[(1- {1-[(1S, 2R, 3R, 4R, 5S) -4- (acetylamino) -2,3-dihydroxy-6,8-dioxabicyclo [3.2. 1] Oct-1-yl] -2,5,8,11-tetraoxatridecan-13-yl} -1H-1,2,3-triazol-4-yl) methoxy] methyl} propan-2-yl ) Amino] -6-oxohexyl} amino) -7-oxoheptanoic acid (67)
7-({6-[(1,3-bis [(1- {1-[(1S, 2R, 3R, 4R, 5S) -4- (acetylamino) in ethanol (2 mL) and tetrahydrofuran (2 mL)]. ) -2,3-bis (acetyloxy) -6,8-dioxabicyclo [3.2.1] oct-1-yl] -2,5,8,11-tetraoxatridecan-13-yl} -1H-1,2,3-triazol-4-yl) methoxy] -2-{[(1- {1-[(1S, 2R, 3R, 4R, 5S) -4- (acetylamino) -2, 3-bis (acetyloxy) -6,8-dioxabicyclo [3.2.1] oct-1-yl] -2,5,8,11-tetraoxatridecan-13-yl} -1H-1 , 2,3-Triazol-4-yl) methoxy] methyl} propan-2-yl) Mino] -6-oxohexyl}} amino) -7-oxoheptanoate ethyl (I-bb-1) (140.0 mg, 0.0675 mmol) in a solution of 5M sodium hydroxide (0.135 mL, .0. 675 mmol) was added. The reaction was stirred overnight at room temperature. The next morning, the reaction was neutralized with acidic resin and filtered. The resin was washed with ethanol and the filtrate was concentrated under reduced pressure. This material was diluted with ethanol (8 mL) and passed through a syringe filter. The filtrate was then concentrated under reduced pressure to give the title compound as a gum (110 mg, 90.8%). Method C: 3-minute run LRMS [1 / 2M + 1 = 898]. ¹ H-NMR (methanol-d ₄ ) δ: 7.9 (s, 3H), 5.21 (d, J = 1.2 Hz, 3H), 4.57-4.62 (m, 6H), 4 .56 (s, 6H), 3.92-3.99 (m, 6H), 3.89 (dd, J = 10.7, 4.9 Hz, 9H), 3.69-3.80 (m, 12H), 3.54-3.68 (m, 42H), 3.12 (t, J = 7.0 Hz, 2H), 2.16 (q, J = 7.3 Hz, 6H), 1.99 ( s, 9H), 1.53-1.67 (m, 6H), 1.49 (dt, J = 14.7, 7.7 Hz, 2H), 1.27-1.41 (m, 4H)

S- [1- (1- {1-[(1S, 2R, 3R, 4R, 5S) -4- (acetylamino) -2,3-dihydroxy-6,8-dioxabicyclo [3.2.1] ] Oct-1-yl] -2,5,8,11-tetraoxatridecan-13-yl} -1H-1,2,3-triazol-4-yl) -4,4-bis {[(1 -{1-[(1S, 2R, 3R, 4R, 5S) -4- (acetylamino) -2,3-dihydroxy-6,8-dioxabicyclo [3.2.1] oct-1-yl] -2,5,8,11-tetraoxatridecan-13-yl} -1H-1,2,3-triazol-4-yl) methoxy] methyl} -6,13-dioxo-2,16,19, 22,25-pentaoxa-5,12-diazaheptaconsan-27-yl] ethanethioe Doo (68)
6-amino-N- (1,3-bis [(1- {1-[(1S, 2R, 3R, 4R, 5S) -4 in N, N-dimethylformamide (1 mL) and tetrahydrofuran (1 mL)]. -(Acetylamino) -2,3-dihydroxy-6,8-dioxabicyclo [3.2.1] oct-1-yl] -2,5,8,11-tetraoxatridecan-13-yl} -1H-1,2,3-triazol-4-yl) methoxy] -2-{[(1- {1-[(1S, 2R, 3R, 4R, 5S) -4- (acetylamino) -2, 3-dihydroxy-6,8-dioxabicyclo [3.2.1] oct-1-yl] -2,5,8,11-tetraoxatridecan-13-yl} -1H-1,2,3 -Triazol-4-yl) methoxy] methyl} propan-2-yl) Xanamide (48) (200.5 mg, 0.117 mmol), S- {15-[(2,5-dioxopyrrolidin-1-yl) oxy] -15-oxo-3,6,9,12-tetraoxa A solution of pentadec-1-yl} ethanethioate (60 mg, 0.14 mmol) and N, N-diisopropylethylamine (0.1 mL, 0.59 mmol) was stirred at room temperature for 20 hours. The reaction mixture was concentrated under reduced pressure. The crude title compound was purified by reverse phase chromatography using the following conditions to give the title compound as a colorless gum (99 mg, 43%). MS [1 / 3M + 1] = 653.7. ¹ H-NMR (methanol-d ₄ ) δ: 8.01 (s, 3H), 5.21 (s, 3H), 4.52-4.64 (m, 12H), 3.95 (t, J = 9.7 Hz, 6H), 3.85-3.91 (m, 9H), 3.74-3.80 (m, 9H), 3.68-3.73 (m, 6H), 3.54 -3.67 (m, 55H), 3.14 (t, J = 7.0 Hz, 2H), 3.06 (t, J = 6.5 Hz, 2H), 2.43 (t, J = 6. 2 Hz, 2H), 2.31 (s, 3H), 2.17 (t, J = 7.3 Hz, 2H), 1.98 (s, 9H), 1.56 (quin, J = 7.5 Hz, 2H), 1.49 (quin, J = 7.3 Hz, 2H), 1.28-1.40 (m, 2H)

Purification conditions The residue was dissolved in dimethyl sulfoxide (1 mL) and purified by reverse phase HPLC. Column: Waters Sunfire C18, 19 × 100, 5u. Mobile phase A: 0.05% TFA in water (v / v). Mobile phase B: acetonitrile (v / v) in 0.05% TFA. From 80.0% H ₂ O / 20.0% acetonitrile in 10.5 minutes to 60.0% H ₂ O / 40.0% acetonitrile with a linear, 60.0% H ₂ O / 40 in 0.5 min Maintain 0% H ₂ O / 100% acetonitrile from 11.0 minutes to 12.0 minutes from 0% acetonitrile to 0% H ₂ O / 100% acetonitrile in a straight line. Flow rate: 25 mL / min.

QC condition:
Column: Waters Atlantis dC18, 4.6 × 50, 5u. Mobile phase A: 0.05% TFA in water (v / v). Mobile phase B: 0.05% TFA in acetonitrile (v / v). 4.0 min from 95.0% H ₂ O / 5.0% acetonitrile to 5% H ₂ O / 95% acetonitrile at a straight line, kept at 5% H ₂ O / 95% acetonitrile to 5.0 min. Flow rate: 2 mL / min.

N- (1,3-bis [(1- {1-[(1S, 2R, 3R, 4R, 5S) -4- (acetylamino) -2,3-dihydroxy-6,8-dioxabicyclo [3 2.1] oct-1-yl] -2,5,8,11-tetraoxatridecan-13-yl} -1H-1,2,3-triazol-4-yl) methoxy] -2- { [(1- {1-[(1S, 2R, 3R, 4R, 5S) -4- (acetylamino) -2,3-dihydroxy-6,8-dioxabicyclo [3.2.1] octa-1 -Yl] -2,5,8,11-tetraoxatridecan-13-yl} -1H-1,2,3-triazol-4-yl) methoxy] methyl} propan-2-yl) -6- { [3- (4-Methyl-2,5-dioxo-2,5-dihydrofuran-3-yl) propano Le] amino} hexanamide (69)
3- (4-Methyl-2,5-dioxo-2,5-dihydrofuran-3-yl) propanoyl chloride (I-bc-1)
(15 mg, 0.074 mmol: see Tetrahedron, 1994, 50, 8969) in dichloromethane (0.1 mL), dichloromethane (0.9 mL), N, N-dimethylformamide (0.1 mL) and anhydrous pyridine ( 6-amino-N- (1,3-bis [(1- {1-[(1S, 2R, 3R, 4R, 5S) -4- (acetylamino)-] in 0.024 mL, 0.30 mmol). 2,3-dihydroxy-6,8-dioxabicyclo [3.2.1] oct-1-yl] -2,5,8,11-tetraoxatridecan-13-yl} -1H-1,2 , 3-Triazol-4-yl) methoxy] -2-{[(1- {1-[(1S, 2R, 3R, 4R, 5S) -4- (acetylamino) -2,3-dihydroxy-6, 8-Geo Sabicyclo [3.2.1] oct-1-yl] -2,5,8,11-tetraoxatridecan-13-yl} -1H-1,2,3-triazol-4-yl) methoxy] methyl } Propan-2-yl) hexanamide acetate (48) (126.0 mg, 0.0736 mmol) was added. The reaction was stirred overnight at room temperature. After 18 hours, an additional 4 equivalents of pyridine (0.024 mL, 0.30 mmol) was added, followed by 1.0 equivalent of acid chloride (I-bc-1) (15 mg, 0.074 mmol). The reaction was stirred overnight at room temperature. The next morning, another 2 equivalents of acid chloride (I-bc-1) was added (30 mg, 0.148 mmol) and the reaction was stirred at room temperature for 4 days. After 4 days, the reaction was concentrated under reduced pressure. The crude title compound was purified by reverse phase chromatography using the following conditions to give the title compound as a gum (16.1 mg, 12%). Method C: 3-minute operation LRMS [1 / 2M + 1 = 910]. ¹ H-NMR (methanol-d ₄ ) δ: 8.00 (s, 3H), 5.21 (s, 3H), 4.52-4.62 (m, 12H), 3.92-3.99 (M, 6H), 3.89 (dd, J = 11.3, 4.7 Hz, 9H), 3.74-3.79 (m, 9H), 3.71 (dd, J = 10.1, 4.3 Hz, 3H), 3.54-3.68 (m, 42H), 3.11 (t, J = 7.0 Hz, 2H), 2.75 (t, J = 7.2 Hz, 2H), 2.46-2.53 (m, 2H), 2.16 (t, J = 7.4 Hz, 2H), 2.05 (s, 3H), 1.99 (s, 9H), 1.55 ( dt, J = 14.9, 7.6 Hz, 2H), 1.46 (quin, J = 7.2 Hz, 2H), 1.24-1.34 (m, 2H)

Purification conditions The residue was dissolved in dimethyl sulfoxide (1 mL) and purified by reverse phase HPLC. Column: Waters Sunfire C18, 19 × 100, 5u. Mobile phase A: 0.05% TFA in water (v / v). Mobile phase B: 0.05% TFA in acetonitrile (v / v). From 75.0% H ₂ O / 25.0% acetonitrile in 10.5 minutes to 65.0% H ₂ O / 35.0% acetonitrile in a straight line, 65.0% H ₂ O / 35 in 0.5 minutes From 0% acetonitrile to 0% H ₂ 0/100% acetonitrile in a straight line, maintain 0% H ₂ 0/100% acetonitrile from 11.0 minutes to 12.0 minutes. Flow rate: 25 mL / min.

QC conditions Column: Waters Atlantis dC18, 4.6 × 50, 5u. Mobile phase A: 0.05% TFA in water (v / v). Mobile phase B: 0.05% TFA in acetonitrile (v / v). Maintain 5% H ₂ 0/95% acetonitrile up to 5.0 minutes from 95.0% H ₂ O / 5.0% acetonitrile in linear to 5% H ₂ 0/95% acetonitrile in 4.0 minutes. Flow rate: 2 mL / min. Retention time = 1.82 minutes. Observed mass = 909.8649. Mass target = 909.2.

tert-Butyl [(5S) -5-({6-[(1,3-bis [(1- {1-[(1S, 2R, 3R, 4R, 5S) -4- (acetylamino) -2, 3-dihydroxy-6,8-dioxabicyclo [3.2.1] oct-1-yl] -2,5,8,11-tetraoxatridecan-13-yl} -1H-1,2,3 -Triazol-4-yl) methoxy] -2-{[(1- {1-[(1S, 2R, 3R, 4R, 5S) -4- (acetylamino) -2,3-dihydroxy-6,8- Dioxabicyclo [3.2.1] oct-1-yl] -2,5,8,11-tetraoxatridecan-13-yl} -1H-1,2,3-triazol-4-yl) methoxy ] Methyl} propan-2-yl) amino] -6-oxohexyl} carbamoyl) -7, 5-Dioxo-37- (pyridin-2-yldisulfanyl) -10,13,16,19,22,25,28,31-octoxa-6,34-diazaheptatricont-1-yl] carbamate (I -Bd-1)
N-6- (tert-butoxycarbonyl) -N-2-[(9H-fluoren-9-ylmethoxy) carbonyl] -L in tetrahydrofuran (2.0 mL) and anhydrous N, N-dimethylformamide (2.0 mL) -Lysine
(70.9 mg, 0.151 mmol) to a solution of 1-hydroxybenzotriazole (24.5 mg, 0.182 mmol) and N- (3-dimethylaminopropyl) -N′-ethylcarbodiimide hydrochloride (35.5 mg 0.182 mmol) was added and the reaction was stirred at room temperature for 1 hour. Starting amine, 6-amino-N- (1,3-bis [(1- {1-[(1S, 2R, 3R, 4R, 5S) -4- (acetylamino) -2,3-dihydroxy-6, 8-dioxabicyclo [3.2.1] oct-1-yl] -2,5,8,11-tetraoxatridecan-13-yl} -1H-1,2,3-triazol-4-yl ) Methoxy] -2-{[(1- {1-[(1S, 2R, 3R, 4R, 5S) -4- (acetylamino) -2,3-dihydroxy-6,8-dioxabicyclo [3. 2.1] oct-1-yl] -2,5,8,11-tetraoxatridecan-13-yl} -1H-1,2,3-triazol-4-yl) methoxy] methyl} propane-2 -Yl) hexanamide (48) (250 mg, 0.151 mmol) The liquid, added as a solid, followed by addition N, N-diisopropylethylamine (0.105 mL, 0.605 mmol) and the reaction was stirred at room temperature for 18 hours. After 18 hours, piperidine (900 mg, 10 mmol, 0.6 mL) was added to the reaction and stirred at room temperature for 3 hours. After 3 hours, the reaction was concentrated under reduced pressure to give a crude gum (450.0 mg, 158%).

tert-Butyl [(5S) -5-({6-[(1,3-bis [(1- {1-[(1S, 2R, 3R, 4R, 5S) -4- (acetylamino) -2, 3-dihydroxy-6,8-dioxabicyclo [3.2.1] oct-1-yl] -2,5,8,11-tetraoxatridecan-13-yl} -1H-1,2,3 -Triazol-4-yl) methoxy] -2-{[(1- {1-[(1S, 2R, 3R, 4R, 5S) -4- (acetylamino) -2,3-dihydroxy-6,8- Dioxabicyclo [3.2.1] oct-yl] -2,5,8,11-tetraoxatridecan-13-yl} -1H-1,2,3-triazol-4-yl) methoxy] methyl } Propan-2-yl) amino] -6-oxohexyl} carbamoyl) -7,35 Dioxo -37- (pyridin-2-yldisulfanyl) -10,13,16,19,22,25,28,31- Okutaokisa -6,34- diaza hept triacontanyl-1-yl] carbamate (70)
Tert-Butyl [(5S) -5-({6-[(1,3-bis [(1- {1-[(1S, 2R]) in N, N-dimethylformamide (1 mL) and tetrahydrofuran (2 mL). , 3R, 4R, 5S) -4- (acetylamino) -2,3-dihydroxy-6,8-dioxabicyclo [3.2.1] oct-1-yl] -2,5,8,11- Tetraoxatridecan-13-yl} -1H-1,2,3-triazol-4-yl) methoxy] -2-{[(1- {1-[(1S, 2R, 3R, 4R, 5S)- 4- (acetylamino) -2,3-dihydroxy-6,8-dioxabicyclo [3.2.1] oct-1-yl] -2,5,8,11-tetraoxatridecan-13-yl } -1H-1,2,3-triazol-4-yl) methoxy] me L} propan-2-yl) amino] -6-oxohexyl} carbamoyl) -7,35-dioxo-37- (pyridin-2-yldisulfanyl) -10,13,16,19,22,25,28, 31-octaoxa-6,34-diazaheptatrita-1-yl] carbamate (I-bd-1) (225 mg, 0.120 mmol), and N- {27-[(2,5-dioxopyrrolidine- 1-yl) -27-oxo-3,6,9,12,15,18,21,24-octaoxaheptakos-1-yl] -3- (pyridin-2-yldisulfanyl) propanamide (111 mg, 0.151 mmol)
To the solution was added N, N-diisopropylethylamine (0.10 mL, 0.574 mmol). The reaction was stirred at room temperature for 3 days. After 3 days, the reaction was concentrated under reduced pressure. A sample of this crude title compound was purified by reverse phase chromatography using the following conditions to give the title compound as a gum (13.8 mg, 4.6%).

Purification conditions The residue was dissolved in dimethyl sulfoxide (1 mL) and purified by reverse phase HPLC. Column: Waters XBridge · C18 19 × 100, 5u. Mobile phase A: 0.03% NH ₄ OH in water (v / v). Mobile phase B: 0.03% NH ₄ OH (v / v) in acetonitrile. 8.5 minutes from 75.0% H ₂ O / 25.0% acetonitrile linearly to 45% H ₂ O / 55% acetonitrile, 0.5 minutes straight from 45% H ₂ O / 55% acetonitrile 0 Maintain 0% H ₂ O / 100% acetonitrile from 9.0 min to 10.0 min to% H ₂ O / 100% MeCN. Flow rate: 25 mL / min.

QC conditions Column: Waters Atlantis dC18, 4.6 × 50, 5u. Mobile phase A: 0.05% TFA in water (v / v). Mobile phase B: 0.05% TFA in acetonitrile (v / v). Maintain 5% H ₂ O / 95% acetonitrile up to 5.0 minutes from 95.0% H ₂ O / 5.0% acetonitrile in 4.0 minutes to 5% H ₂ O / 95% acetonitrile in a straight line. Flow rate: 2 mL / min. Retention time = 2.06 minutes. Observed mass = 843.5669, mass target = 833.4

(33S) -33- (4-aminobutyl) -N- (1,3-bis [(1- {1-[(1S, 2R, 3R, 4R, 5S) -4- (acetylamino) -2, 3-dihydroxy-6,8-dioxabicyclo [3.2.1] oct-1-yl] -2,5,8,11-tetraoxatridecan-13-yl} -1H-1,2,3 -Triazol-4-yl) methoxy] -2-{[(1- {1-[(1S, 2R, 3R, 4R, 5S) -4- (acetylamino) -2,3-dihydroxy-6,8- Dioxabicyclo [3.2.1] oct-1-yl] -2,5,8,11-tetraoxatridecan-13-yl} -1H-1,2,3-triazol-4-yl) methoxy ] Methyl} propan-2-yl) -3,31,34-trioxo-1- (pyridine-2) Yldisulfanyl) -7,10,13,16,19,22,25,28- Okutaokisa -4,32,35- triazacyclononane hept triacontanyl -41- amide (71)
The remaining crude tert-butyl [(5S) -5-({6-[(1,3-bis [(1- {1-[(1S, 2R, 3R, 4R, 5S) -4- (acetylamino) -2,3-dihydroxy-6,8-dioxabicyclo [3.2.1] oct-1-yl] -2,5,8,11-tetraoxatridecan-13-yl} -1H-1, 2,3-triazol-4-yl) methoxy] -2-{[(1- {1-[(1S, 2R, 3R, 4R, 5S) -4- (acetylamino) -2,3-dihydroxy-6 , 8-dioxabicyclo [3.2.1] oct-yl] -2,5,8,11-tetraoxatridecan-13-yl} -1H-1,2,3-triazol-4-yl) Methoxy] methyl} propan-2-yl) amino] -6-oxohexyl} carbamoyl) 7,35-Dioxo-37- (pyridin-2-yldisulfanyl) -10,13,16,19,22,25,28,31-octoxa-6,34-diazaheptatricont-1-yl] carbamate (70) was diluted in methanol (3 mL), 4.0 M hydrogen chloride in dioxane (0.898 mL, 3.59 mmol) was added and the reaction was stirred at room temperature for 18 hours. After 18 hours, the reaction was concentrated under reduced pressure. The crude title compound was purified by reverse phase chromatography using the following conditions to give the title compound as a gum (78.9 mg, 27%).

Purification conditions The residue was dissolved in dimethyl sulfoxide (1 mL) and purified by reverse phase HPLC. Column: Waters Sunfire C18, 19 × 100, 5u. Mobile phase A: 0.05% TFA in water (v / v). Mobile phase B: 0.05% TFA in acetonitrile (v / v). 8.5 minutes from 80.0% H ₂ O / 20.0% acetonitrile to 45% H ₂ O / 55% acetonitrile in a straight line, 0.5 minutes from 45% H ₂ O / 55% acetonitrile in a straight line to 0% Maintain 0% H ₂ O / 100% acetonitrile from 9.0 to 10.0 minutes to% H ₂ O / 100% MeCN. Flow rate: 25 mL / min.

QC conditions Column: Waters Atlantis dC18, 4.6 × 50, 5u. Mobile phase A: 0.05% TFA in water (v / v). Mobile phase B: 0.05% TFA in acetonitrile (v / v). Maintain 5% H ₂ O / 95% acetonitrile up to 5.0 minutes from 95.0% H ₂ O / 5.0% acetonitrile in 4.0 minutes to 5% H ₂ O / 95% acetonitrile in a straight line. Flow rate: 2 mL / min. Retention time = 1.7544 minutes. Observed mass = 601.159 (1 / 4M + 1). Mass target = 600.3)

LCMS method E: ESI (m / z) using MaxEnt deconvolution software.
Column: Acquity / BEH300 / C41.7um
Mobile phase: A = 0.1% formic acid in water. B = 0.1% formic acid in ACN Gradient: 97% A to 5% A in 2 minutes. Maintain at 5% A for 0.75 minutes. Thereafter, the process returns to the start condition.
Temperature: 70 ° C
MS detection = ESI 0-2000 Dalton, MaxEnt is used to analyze higher MW species.

9H-Fluoren-9-ylmethyl [(2S) -1-({6- [1,3-bis (1- {1-[(1S, 2R, 6R, 7R, 8S) -7- (acetylamino)- 4,4-Dimethyl-3,5,9,11-tetraoxatricyclo [6.2.1.0-2,6-]-undec-1-yl] -2,5,8,11-tetraoxatri Decan-13-yl} -1H-1,2,3-triazol-4-yl) methoxy] -2-{[(1- {1-[(1S, 2R, 6R, 7R, 8S) -7- ( Acetylamino) -4,4-dimethyl-3,5,9,11-tetraoxatricyclo [6.2.1.0-2,6-]-undec-1-yl] -2,5,8,11 -Tetraoxatridecan-13-yl} -1H-1,2,3-triazol-4-yl) methoxy] methyl} Pan 2-yl) amino] -6-oxo-hexyl} amino) -1-Okisopenta-4-yn-2-yl] carbamate (I-be-1)
In a vial, (2S) -2-{[(9H-fluoren-9-ylmethoxy) carbonyl] amino} pent-4-ynoic acid (51.7 mg, 0.154 mmol), HBTU (58.0 mg, 0.18 mmol) And anhydrous N, N-dimethylformamide (0.5 ml) and tetrahydrofuran (0.5 ml) were added followed by N, N-diisopropylethylamine (0.10 ml, 0.593 mmol) and the mixture was stirred for 5 minutes. . This mixture was added to 6-amino-N- (1,3-bis [(1- {1-[(1S, 2R, 6R, 7R, 8S)] in anhydrous N, N-dimethylformamide (0.75 mL). -7- (acetylamino) -4,4-dimethyl-3,5,9,11-tetraoxatricyclo [6.2.1.0-2,6-] undec-1-yl] -2,5 , 8,11-tetraoxatridecan-13-yl} -1H-1,2,3-triazol-4-yl) methoxy] -2-{[(1- {1-[(1S, 2R, 6R, 7R, 8S) -7- (acetylamino) -4,4-dimethyl-3,5,9,11-tetraoxatricyclo [6.2.1.0-2,6-]-undec-1-yl] -2,5,8,11-tetraoxatridecan-13-yl} -1H-1,2,3-triazole- - yl) methoxy] methyl} propan-2-yl) hexanamide (1-ag-1) (210mg, was added dropwise to a solution of 0.119 mmol). Stir the reaction at room temperature until the reaction is complete by LCMS. Concentrated to crude oil using Gene vac and purified on silica gel column using MeOH / DCM gradient from 0% to 20%. The isolated fractions were concentrated to give 205 mg (82% yield) of the title compound as a white form. Method C: LRMS (1 / 2M + 1 = 1045.7). The NMR spectrum showed rotamers (1: 4 ratio) reported as partial hydrogen.
¹ H-NMR (methanol-d ₄ ) δ: 7.96 (s, 3H), 7.85 (d, J = 8.2 Hz, 0.4H), 7.79 (d, J = 7.6 Hz, 1.6H), 7.73 (d, J = 8.2 Hz, 0.4H), 7.66 (d, J = 7.0 Hz, 1.6H), 7.51 (t, J = 7.6 Hz) , 0.4H), 7.43-7.47 (m, J = 7.6 Hz, 0.4H), 7.39 (t, J = 7.3 Hz, 1.6H), 7.28-7. 32 (m, 1.6H), 5.22 (s, 3H), 4.50-4.62 (m, 12H), 4.38-4.45 (m, 1H), 4.30-4. 36 (m, 1H), 4.28 (d, J = 5.9 Hz, 3H), 4.19-4.25 (m, 2H), 4.15 (t, J = 6.2 Hz, 3H), 3.88-3. 4 (m, 6H), 3.87 (t, J = 4.7 Hz, 6H), 3.82 (d, J = 7.6 Hz, 3H), 3.72-3.78 (m, 12H), 3.48-3.70 (m, 36H), 3.12-3.20 (m, 2H), 2.65 (br.s., 1H), 2.54-2.61 (m, 1H) , 2.40 (br.s., 1H), 2.15 (t, J = 6.7 Hz, 2H), 1.97 (s, 9H), 1.47 (s, 9H), 1.43- 1.59 (m, 4H), 1.31 (s, 9H), 1.36 (s, 2H)

tert-Butyl (4S) -5-({6-[(1,3-bis [(1- {1-[(1S, 2R, 6R, 7R, 8S) -7- (acetylamino) -4,4 -Dimethyl-3,5,9,11-tetraoxatricyclo [6.2.1.0-2,6-]-undec-1-yl] -2,5,8,11-tetraoxatridecan-13 -Yl} -1H-1,2,3-triazole-4-yl) methoxy] -2-{[(1- {1-[(1S, 2R, 6R, 7R, 8S) -7- (acetylamino) -4,4-dimethyl-3,5,9,11-tetraoxatricyclo [6.2.1.0-2,6-]-undec-1-yl] -2,5,8,11-tetraoxa Tridecan-13-yl} -1H-1,2,3-triazol-4-yl) methoxy] methyl} propan-2-y ) Amino] -6-oxo-hexyl} amino) -4 - {[(9H- fluoren-9-ylmethoxy) carbamoyl] amino} -5-oxopentanoate (I-bf-1)
The above title compound was synthesized in the same manner as (I-be-1), and (2S) -5-tert-butoxy-2-{[(9H-fluoren-9-ylmethoxy) carbonyl] amino} -5 -Oxopentanoic acid (63.1 mg, 0.148 mmol) was used to give 204 mg (82% yield) of the title compound in white form. Method C: LRMS (1 / 2M + 1 = 1090.7). The NMR spectrum showed rotamers (1: 4 ratio), which is reported as partial hydrogen. ¹ H-NMR (METHANOL-d ₄ ) δ: 7.96 (s, 3H), 7.86 (d, J = 8.2 Hz, 0.4H), 7.80 (d, J = 7.6 Hz, 1.6H), 7.73 (d, J = 8.2 Hz, 0.4H), 7.66 (t, J = 6.5 Hz, 1.6H), 7.51 (t, J = 7.6 Hz) , 0.4H), 7.43-7.47 (m, J = 8.2 Hz, 0.4H), 7.39 (t, J = 7.3 Hz, 1.6H), 7.28-7. 33 (m, 1.6H), 5.23 (s, 3H), 4.50-4.61 (m, 12H), 4.38-4.46 (m, 1H), 4.31-4. 38 (m, 1H), 4.28 (d, J = 5.9 Hz, 3H), 4.22 (t, J = 6.7 Hz, 1H), 4.15 (t, J = 6.2 Hz, 3H) ), 4 04-4.11 (m, 1H), 3.85-3.96 (m, 12H), 3.82 (d, J = 7.6 Hz, 3H), 3.71-3.78 (m, 12H) ), 3.51-3.70 (m, 36H), 3.10-3.19 (m, 2H), 2.29 (t, J = 7.3 Hz, 2H), 2.15 (t, J = 7.0 Hz, 2H), 2.01-2.10 (m, 1H), 1.98 (s, 9H), 1.78-1.90 (m, 1H), 1.51-1.59. (M, 2H), 1.47 (s, 11H), 1.44 (s, 9H), 1.32 (s, 11H)

9H-Fluoren-9-ylmethyl [(2S) -3-azido-1-({6-[(1,3-bis [(1- {1-[(1S, 2R, 6R, 7R, 8S) -7 -(Acetylamino) -4,4-dimethyl-3,5,9,11-tetraoxatricyclo [6.2.1.0-2,6-]-undec-1-yl] -2,5,8 , 11-tetraoxatridecan-13-yl} -1H-1,2,3-triazol-4-yl) methoxy] -2-{[(1- {1-[(1S, 2R, 6R, 7R, 8S) -7- (acetylamino) -4,4-dimethyl-3,5,9,11-tetraoxatricyclo [6.2.1.0-2,6-] undec-1-yl] -2 , 5,8,11-tetraoxatridecan-13-yl} -1H-1,2,3-triazol-4-yl) meth Shi] methyl} propan-2-yl) amino] -6-oxo-hexyl} amino) -1-oxo-2-yl] carbamate (I-bg-1)
The above title compound was synthesized in the same manner as (I-be-1), and 3-azido-N-[(9H-fluoren-9-ylmethoxy) carbonyl] -L-alanine (51.6 mg, 0.148 mmol) 191 mg (68% yield) of the title compound was obtained in white form. Method C: LRMS (1 / 2M + 1 = 1054.1). The NMR spectrum showed rotamers (1: 4 ratio) reported as partial hydrogen. ¹ H-NMR (methanol-d ₄ ) δ: 7.97 (s, 3H), 7.85 (d, J = 8.2 Hz, 0.4H), 7.80 (d, J = 7.0 Hz, 1.6H), 7.73 (d, J = 8.2 Hz, 0.4H), 7.67 (d, J = 7.0 Hz, 1.6H), 7.50 (d, J = 7.6 Hz) , 0.4H), 7.45 (d, J = 7.6 Hz, 0.4H), 7.39 (t, J = 7.3 Hz, 1.6H), 7.29-7.33 (m, 1.6H), 5.22 (s, 3H), 4.52-4.62 (m, 12H), 4.41-4.47 (m, 1H), 4.34-4.40 (m, 1H), 4.28 (d, J = 5.9 Hz, 3H), 4.21-4.26 (m, 1H), 4.15 (t, J = 6.5 Hz, 3H), 3.89- 3.95 (m, 6H), 3.87 (t, J = 5.0 Hz, 6H), 3.82 (d, J = 7.6 Hz, 3H), 3.72-3.79 (m, 12H), 3.49- 3.69 (m, 39H), 3.06-3.23 (m, 2H), 2.13-2.23 (m, 2H), 1.98 (s, 9H), 1.50-1. 63 (m, 4H), 1.47 (s, 9H), 1.32 (s, 9H), 1.28-1.40 (m, 2H)

9H-Fluoren-9-ylmethyl [(2S) -1-({6-[(1,3-bis [(1- {1-[(1S, 2R, 6R, 7R, 8S) -7- (acetylamino ) -4,4-dimethyl-3,5,9,11-tetraoxatricyclo [6.2.1.0-2,6-]-undec-1-yl] -2,5,8,11-tetra Oxatridecan-13-yl} -1H-1,2,3-triazol-4-yl) methoxy] -2-{[(1- {1-[(1S, 2R, 6R, 7R, 8S) -7 -(Acetylamino) -4,4-dimethyl-3,5,9,11-tetraoxatricyclo [6.2.1.0-2,6-]-undec-1-yl] -2,5,8 , 11-Tetraoxatridecan-13-yl} -1H-1,2,3-triazol-4-yl) methoxy] methyl Propan-2-yl) amino] -6-oxohexyl} amino) -3- (1- {15-[(2,5-dioxopyrrolidin-1-yl) oxy] -15-oxo-3,6, 9,12-tetraoxapentadec-1-yl} -1H-1,2,3-triazol-4-yl) -1-oxopropan-2-yl] carbamate (I-bh-1)
Example alkyne (I-be-1) (36.5 mg, 0.0175 mmol), 1-[(1-azido-15-oxo-3,6,9,12-tetraoxapentadecan-15-yl) oxy Pyrrolidine-2,5-dione (11 mg, 0.0283 mmol) and sodium ascorbate (4.0 mg, 0.020 mmol) were added into tert-butanol (1 ml) to obtain a clear solution. A solution of freshly prepared copper sulfate (0.93 mg, 0.0058 mmol) in water (0.4 ml) is added to the reaction flask. After stirring for 1 hour at room temperature, the reaction became creamy and then turned light green / blue. After 1 hour, the reaction was extracted 3 times with dichloromethane (2 ml). The combined organic layers were dried over anhydrous sodium sulfate and concentrated to give a crude solid. Method E: M / Z = 2478.0

S- {15-[(2S, 4R) -2-{[Bis (4-methoxyphenyl) (phenyl) methoxy] methyl} -4-hydroxypyrrolidin-1-yl] -15-oxo-3,6,9 , 12-Tetraoxapentadec-1-yl} ethanethioate (I-bi-1)
(3R, 5S) -5-{[Bis (4-methoxyphenyl) (phenyl) methoxy] methyl} pyrrolidin-3-ol was prepared according to published procedures (Prakash, TP et al., 8996-8807, Nucleic Acids Res., 2014, 42, 8796. (3R, 5S) -5-{[Bis (4-methoxyphenyl) (phenyl) in N, N-dimethylformamide (1 mL) and tetrahydrofuran (1 mL). ) Methoxy] methyl} pyrrolidin-3-ol (0.18 g, 0.43 mmol), S-{[(2,5-dioxopyrrolidin-1-yl) oxy] -15-oxo-3,6,9, 12-tetraoxapentadec-1-yl} ethanethioate (0.20 g, 0.48 mmol) and N, N-diisopropyl A solution of ruethylamine (0.28 g, 2.2 mmol) was stirred for 5 days at room temperature The reaction mixture was diluted with water (10 mL) and extracted with ethyl acetate (3 × 5 mL). Dry over sodium sulfate, filter and concentrate The crude residue was purified by column chromatography (0% to 7% methanol in dichloromethane) to give the title compound as a colorless oil (0.19 g). 62%).

  Method F: 3.0 minute run LRMS (low resolution mass spectrometry): Waters Acquity UPLC BEH, 2.1 mm x 50 mm, C18 1.8 μm. Column temperature 60 ° C. Mobile phase A: 0.1% ammonia in water (v / v). Mobile phase B: 0.1% ammonia in acetonitrile (v / v). Flow rate = 1.25 mL / min. Initial conditions: A-95%: B-5%. The initial condition is maintained for 0.0 to 0.1 minutes. 0.1-2.6 minutes increased to A-5%: B-95%. From 2.6 to 2.95 minutes, A-5%: B-95% was maintained. 2.95-3.0 minutes return to initial conditions.

Method D: Three-minute LRMS [M + 1 = 726.6]. The NMR spectra were confused by the presence of rotamers in the 2: 1 mixture, but are reported as seen. ¹ H-NMR (600 MHz, methanol-d ₄ ) δ: 7.34-7.42 (m, 2H), 7.23-7.32 (m, 6H), 7.16-7.23 (m, 1H), 6.81-6.90 (m, 4H), 4.52-4.62 (m, 0.66H), 4.48 (dt, J = 9.7, 5.1 Hz, 0.33H) ), 4.22-4.34 (m, 1H), 3.67-3.84 (m, 8H), 3.40-3.64 (m, 16H), 3.22 (m, J = 5) .6, 5.6 Hz, 0.66 H), 3.13 (dd, J = 9.4, 2.3 Hz, 0.66 H), 3.01-3.10 (m, 1.66 H), 54-2.68 (m, 1.66H), 2.26-2.37 (m, 3.33H), 2.17-2.25 (m, 1H), 2.02-2.11 (m 0.33H), 1.91-2.00 (m, 0.66H).

N- {6-[(1,3-bis [(1- {1-[(1S, 2R, 3R, 4R, 5S) -4- (acetylamino) -2,3-dihydroxy-6,8-di Oxabicyclo [3.2.1] oct-1-yl] -2,5,8,11-tetraoxatridecan-13-yl} -1H-1,2,3-triazole-4-yl) methoxy] -2-{[(1- {1-[(1S, 2R, 3R, 4R, 5S) -4- (acetylamino) -2,3-dihydroxy-6,8-dioxabicyclo [3.2.1 ] Oct-1-yl] -2,5,8,11-tetraoxatridecan-13-yl} -1H-1,2,3-triazol-4-yl) methoxy] methyl} propan-2-yl) Amino] -6-oxohexyl} -7-[(2S, 4R) -2-{[bis (4-methoxy) Eniru) (phenyl) methoxy] methyl} -4-hydroxy-1-yl] -7-oxo-heptanamide (72)
Of (3R, 5S) -5-{[bis (4-methoxyphenyl) (phenyl) methoxy] methyl} pyrrolidin-3-ol (14 mg, 0.034 mmol) in N, N-dimethylformamide (0.1 mL). The solution was dissolved in 7-({6-[(1,3-bis [(1- {1-[(1S, 2R, 3R, 4R, 5S) -4-] in N, N-dimethylformamide (1 mL). (Acetylamino) -2,3-bis (acetyloxy) -6,8-dioxabicyclo [3.2.1] oct-1-yl] -2,5,8,11-tetraoxatridecane-13 -Yl} -1H-1,2,3-triazol-4-yl) methoxy] -2-{[(1- {1-[(1S, 2R, 3R, 4R, 5S) -4- (acetylamino) -2,3-bis (acetyloxy) -6,8-dioxa Cyclo [3.2.1] oct-1-yl] -2,5,8,11-tetraoxatridecan-13-yl} -1H-1,2,3-triazol-4-yl) methoxy] methyl } Propan-2-yl) amino] -6-oxohexyl}} amino) -7-oxoheptanoate (I-bb-1) (61 mg, 0.034 mmol), TBTU (16 mg, 0.049 mmol) and N , N-diisopropylethylamine (22 mg, 0.17 mmol) was added and the reaction was stirred at room temperature for 5 days. (3R, 5S) -5-{[Bis (4-methoxyphenyl) (phenyl) methoxy] methyl} pyrrolidin-3-ol (14 mg, 0.034 mmol), TBTU (16 mg, 0.049 mmol) and N, N-diisopropylethylamine (22 mg, 0.17 mmol) was added and the reaction was stirred for another 2 days. The reaction mixture was concentrated under reduced pressure and purified under the following conditions.

Purification conditions The residue was dissolved in dimethyl sulfoxide (1 mL) and purified by reverse phase HPLC. Column: Waters XBridge · C18, 19 × 100, 5u. Mobile phase A: 0.03% NH ₄ OH in water (v / v). Mobile phase B: 0.03% NH ₄ OH (v / v) in acetonitrile.
From 60.0% H ₂ O / 40.0% acetonitrile in 10.5 minutes to 40% H ₂ 0/60% acetonitrile in a straight line, maintaining 0% H ₂ 0/100% acetonitrile until 12.0 minutes. Flow rate: 25 mL / min.
The title compound was obtained as a colorless residue (1 mg, 1%). Method F: 3-minute LRMS [1 / 2M-1] = 1097.2. ¹ H-NMR (600 MHz, methanol-d ₄ ) δ: 7.9 (s, 3H), 7.40 (d, J = 7.6 Hz, 2H), 7.24-7.32 (m, 6H) 7.16-7.22 (m, 1H), 6.85 (d, J = 8.8 Hz, 4H), 5.21 (s, 3H), 4.52-4.64 (m, 12H) , 4.39-4.47 (m, 1H), 4.21 (br.s., 1H), 3.95 (t, J = 9.7 Hz, 6H), 3.85-3.92 (m , 9H), 3.44-3.83 (m, 65H), 3.09-3.17 (m, 2H), 2.24-2.49 (m, 2H), 2.12-2.23 (M, 4H), 1.93-2.11 (m, 10H), 1.53-1.70 (m, 6H), 1.44-1.52 (m, 2H), 1.22-1 42 (m, 4H)

Scheme 9 outlines a general procedure that can be used to provide compounds of the invention. In Step 1 and Step 4 of Scheme 9, Carbohydrate Research, 135, 53 (1984). Paulsen and M.M. Synthetic intermediates (I-bj) that can be prepared by the procedure described by Paal are suitable Lewis acids (eg, as described in Takao Aoki et.al., WO / 20060129129, March 16, 2006). (I-bk) by glycosylation (using protocols well known to those skilled in the art, such as using a suitable organic solvent such as dichloromethane, diethyl ether boron trifluoride complex), dichloromethane, and a suitable alcohol). And intermediates such as (I-bn) can be prepared. In Step 2 and Step 5 of Scheme 9, intermediates (I-bk) and (I−) in the presence of an alkoxide in a solvent or solvent mixture (eg, sodium methoxide) at a temperature of about 0 ° C. to room temperature. Treatment of bn) gives intermediates such as (I-bl) and (I-bo). In Step 3 of Scheme 9, the secondary hydroxyl group in a compound such as (I-b1) is converted to a suitable protecting group (eg, N, N-dimethylformamide at a temperature in the range of about room temperature to about 90 ° C.). Is further protected as a cyclic ketal during treatment with 2,2′-dimethoxypropane under acidic conditions in a solvent such as (I-bm). In addition, in step 6 of Scheme 9, compound (I-bo) is a reducing agent known to reduce an azide group to the corresponding amine (eg, transition metal mediated catalytic hydrogenation, a classic well known to those skilled in the art). Use of triphenylphosphine in water under typical experimental conditions). Subsequent treatment in the presence of an acylating agent (eg acetyl chloride in a solvent such as dichloromethane or tetrahydrofuran in the presence of acetic anhydride or pyridine or triethylamine). When a alkoxide (eg, sodium methoxide) is subsequently added in a solvent, or a mixture of solvents such as alcohol solvent or tetrahydrofuran, at a temperature in the range of about 0 ° C. to room temperature, a compound such as (I-bp) can get. In Step 7 of Scheme 9, the secondary hydroxyl group in compound (I-bp) is converted to a suitable protecting group (such as N, N-dimethylformamide at a temperature ranging from about room temperature to about 90 ° C). Further protection by a treatment with 2,2-dimethoxypropane under acidic conditions in a solvent (as a cyclic ketal) can give intermediates such as (I-bq). Then, using synthetic transformations and functional and protecting group manipulations well known to those skilled in the art similarly described for (Id) and (Ie) are used for (I-bm) and (I-bq). Which can be combined with the desired linker X and ligand Y of interest to further functionalize and derivatize the primary hydroxyl group to form an XY-containing compound as claimed in the present invention. Prepared. Removal of the protecting group (eg, Pg) using reagents and conditions well known to those skilled in the art (eg, when two Pg forms a cyclic ketal such as acetonide, it is acetic acid, alcohol solvent, water, XY-containing compounds claimed in the present invention can be removed under acidic conditions using acetic acid or the like acid in a mixture of solvents such as tetrahydrofuran, at temperatures between room temperature and about 80 ° C., exposing secondary hydroxyl groups Lead. For example, alkylation of the primary hydroxyl group in (I-bq) can result in the corresponding ether-linked XY-containing compound claimed in the present invention after manipulation and removal of the protecting group. Ester-linked, carbonate-linked and carbamate-linked XY-containing compounds claimed in the present invention can also be prepared using (I-bm) or (I) using appropriate reactants and reagents well known to those skilled in the art. It can be conveniently obtained from an intermediate such as -bq). Further manipulations on (I-bq) similar to those described above for (I-e) are followed by intermediates (III-e-1) to (III-e-11) and (IV-e-1) In a manner similar to those skilled in the art for (IV-e-9), (III-bo-1) to (III-bo-22) and (IV-bo-1) to (IV-bo-1) Makes it possible to obtain such intermediates (Ie).

2- {2- [2- (2-azidoethoxy) ethoxy] ethoxy} ethyl 3,4,6-tri-O-acetyl-2- (acetylamino) -2-deoxy-β-D-galactopyranoside (I-br-1)
2- {2- [2- (2-azidoethoxy) ethoxy] ethoxy} ethyl 2- (acetylamino) -2-deoxy-β-D-galactopyranoside (WO 2006 / 028129A1, March 16, 2006: 1600 mg, 3.78 mmol) was dissolved in anhydrous pyridine (3.0 mL, 38 mmol), to which acetic anhydride (5.36 mL, 56.8 mmol) was added at room temperature. The reaction was then heated at 50 ° C. overnight. The next morning, the reaction was concentrated under reduced pressure. The crude material was purified using CombiFlash Rf (RediSep 40 g silica gel column) eluting with a methanol / dichloromethane gradient from 0% to 20% to give the title compound as a gum (1800 mg, 87 %). 3-minute operation LRMS [M + 1 = 549.3]

Benzyl (6-{[1,3-bis {[1- (2- {2- [2- (2-{[3,4,6-tri-O-acetyl-2- (acetylamino) -2- Deoxy-β-D-galactopyranosyl] oxy} ethoxy) ethoxy] ethoxy} ethyl) -1H-1,2,3-triazol-4-yl] methoxy} -2-({[1- (2- { 2- [2- (2-{[3,4,6-tri-O-acetyl-2- (acetylamino) -2-deoxy-β-D-galactopyranosyl] oxy} ethoxy) ethoxy] ethoxy} Ethyl) -1,2,3-triazol-4-yl] methoxy} methyl) propan-2-yl] amino} -6-oxohexyl) carbamate (I-bs-1)
A 50 mL round bottom flask equipped with a stir bar was charged with (Iq-1) (250 mg, 0.518 mmol) and 2- {2- [2- (2-azidoethoxy) in t-butanol (7 mL). Ethoxy] ethoxy} ethyl 3,4,6-tri-O-acetyl-2- (acetylamino) -2-deoxy-β-D-galactopyranoside (I-br-1) (750 mg, 1.37 mmol) Followed by water (3 mL), sodium ascorbate (1050 mg, 5.18 mmol) was added undiluted and the reaction was purged with nitrogen for 30 minutes. Copper sulfate (83.5 mg, 0.518 mmol) was added to 0.5 mL of water (deionized water) and stirred at room temperature for 24 hours. After 24 hours, the reaction mixture was added to saturated aqueous ammonium chloride (20 mL) and concentrated ammonium hydroxide (2 mL) to quench the reaction and extracted three times with dichloromethane (10 mL). The combined organic layers were dried over magnesium sulfate, filtered and concentrated under reduced pressure. The crude material was purified using CombiFlash Rf (RediSep 40 g Gold silica gel column) eluting with a methanol / dichloromethane gradient from 0% to 20% to give the title compound as a gum. (574.0 mg, 52.1%). 3-minute operation LRMS [1 / 2M + 1 = 1065.2]

6-amino-N- [1,3-bis {[1- (2- {2- [2- (2-{[2- (acetylamino) -2-deoxy-β-D-galactopyranosyl] Oxy} ethoxy) ethoxy] ethoxy} ethyl) -1H-1,2,3-triazol-4-yl] methoxy} -2-({[1- (2- {2- [2- (2-{[2 -(Acetylamino) -2-deoxy-β-D-galactopyranosyl] oxy} ethoxy) ethoxy] ethoxy} ethyl) -1H-1,2,3-triazol-4-9yl] methoxy} methyl) propane -2-yl] hexanamide (I-bt-1)
Benzyl (6-{[1,3-bis {[1- (2- {2- [2- (2-{[3,4,6-tri-O] in methanol (10 mL) and tetrahydrofuran (4 mL). -Acetyl-2- (acetylamino) -2-deoxy-β-D-galactopyranosyl] oxy} ethoxy) ethoxy] ethoxy} ethyl) -1H-1,2,3-triazol-4-yl] methoxy} -2-({[1- (2- {2- [2- (2-{[3,4,6-tri-O-acetyl-2- (acetylamino) -2-deoxy-β-D-galacto Pyranosyl] oxy} ethoxy) ethoxy] ethoxy} ethyl) -1,2,3-triazol-4-yl] methoxy} methyl) propan-2-yl] amino} -6-oxohexyl) carbamate (I-bs) -1) (274.0 mg, 0. 129 mmol) solution was added 25 wt% sodium methoxide in methanol (0.412 mL, 1.80 mmol) and the reaction was stirred at room temperature for 20 hours. After 20 hours, Amberlite IR-120 (H) and an ion exchange resin (CAS # 7892-04-0, rinsed 3 times with methanol) were added until pH = about 6. The mixture was filtered and the resin was washed with methanol (2 × 15 mL). The filtrate was concentrated under reduced pressure to give the title compound as a gum (243.0 mg, 108%). 3-minute operation LRMS [M + 1 = 1751.4]

6-amino-N- [1,3-bis {[1- (2- {2- [2- (2-{[2- (acetylamino) -2-deoxy-β-D-galactopyranosyl] Oxy} ethoxy) ethoxy] ethoxy} ethyl) -1H-1,2,3-triazol-4-yl] methoxy} -2-({[1- (2- {2- [2- (2-{[2 -(Acetylamino) -2-deoxy-β-D-galactopyranosyl] oxy} ethoxy) ethoxy] ethoxy} ethyl) -1H-1,2,3-triazol-4-yl] methoxy} methyl) propane- 2-yl] hexaneamide acetate (I-bu-1)
6-Amino-N- [1,3-bis {[1- (2- {2- [2- (2-{[] in methanol (13.9 mL) and acetic acid (0.01 mL, 0.2 mmol). 2- (acetylamino) -2-deoxy-β-D-galactopyranosyl] oxy} ethoxy) ethoxy] ethoxy} ethyl) -1H-1,2,3-triazol-4-yl] methoxy} -2- ({[1- (2- {2- [2- (2-{[2- (acetylamino) -2-deoxy-β-D-galactopyranosyl] oxy} ethoxy) ethoxy] ethoxy} ethyl)- 1H-1,2,3-triazol-4-9yl] methoxy} methyl) propan-2-yl] hexanamide (I-bt-1) (243 mg, 0.139 mmol) was added to 10% palladium on carbon ( H queue using small cartridge) The tube was passed using the following parameters (temperature = 50 ° C., flow rate = 1.0 mL / min, pressure = total H ₂ (1 bar)). The solution was collected and concentrated under reduced pressure to give the title compound as a gum (187 mg, 80%).

N- [1,3-bis {[1- (2- {2- [2- (2-{[2- (acetylamino) -2-deoxy-β-D-galactopyranosyl] oxy} ethoxy) Ethoxy] ethoxy} ethyl) -1H-1,2,3-triazol-4-yl] methoxy} -2-({[1- (2- {2- [2- (2-{[2- (acetylamino) ) -2-Deoxy-β-D-galactopyranosyl] oxy} ethoxy) ethoxy] ethoxy} ethyl) -1H-1,2,3-triazol-4-yl] methoxy} methyl) propan-2-yl] -3,31-dioxo-1- (pyridin-2-yldisulfanyl) -7,10,13,16,19,22,25,28-octoxa-4,32-diazaoctatriacontane 38-amide (73 )
6-amino-N- [1,3-bis {[1- (2- {2- [2- (2-{[2- (2-mL)] in N, N-dimethylformamide (1 mL) and tetrahydrofuran (1 mL). Acetylamino) -2-deoxy-β-D-galactopyranosyl] oxy} ethoxy) ethoxy] ethoxy} ethyl) -1H-1,2,3-triazol-4-yl] methoxy} -2-({[ 1- (2- {2- [2- (2-{[2- (acetylamino) -2-deoxy-β-D-galactopyranosyl] oxy} ethoxy) ethoxy] ethoxy} ethyl) -1H-1 , 2,3-Triazol-4-yl] methoxy} methyl) propan-2-yl] hexaneamide acetate (I-bu-1) (78.8 mg, 0.047 mmol) and N- {27-[(2, 5-Dioxopyrrolidine -1-yl) oxy] -27-oxo-3,6,9,12,15,18,21,24-octaoxaheptakos-1-yl-3- (pyridin-2-yldisulfanyl) propanamide
To a solution of (MFCD13185003: 41.5 mg, 0.0564 mmol), N, N-diisopropylethylamine (0.04 mL, 0.2 mmol) was added. The reaction was stirred at room temperature for 18 hours. After 18 hours, the reaction was concentrated under reduced pressure. The crude title compound was purified by reverse phase chromatography using the following conditions to give the title compound as a gum (42.7 mg, 41%).

Purification conditions The residue was dissolved in dimethyl sulfoxide (1 mL) and purified by reverse phase HPLC. Column: Waters Sunfire C18, 19 × 100, 5μ. Mobile phase A: 0.05% TFA in water (v / v). Mobile phase B: 0.05% TFA in acetonitrile (v / v). Gradient: 10.5 minutes 80.0% H ₂ O / 20.0% acetonitrile to 65.0% H ₂ O / 35.0% acetonitrile with a linear, 65.0% H ₂ O with 0.5 minutes from /35.0Pasento acetonitrile to 0% H ₂ O / 100% acetonitrile linear, maintain 0% H ₂ O / 100% acetonitrile to 11.0 minutes 12.0 minutes. Flow rate: 25 mL / min.

QC condition:
Column: Waters Atlantis dC18, 4.6 × 50, 5μ. Mobile phase A: 0.05% TFA in water (v / v). Mobile phase B: 0.05% TFA in acetonitrile (v / v). Gradient: 4.0 min from 95.0% H ₂ O / 5.0% acetonitrile to 5% H ₂ O / 95% acetonitrile at a straight line, maintaining a 5% H ₂ O / 95% acetonitrile to 5.0 min . Flow rate: 2 mL / min, retention time = 1.6667 min, observed mass = 746.5042, mass target = 745.3).

Peptide Experimental Procedures Peptide purity check: Unless otherwise stated, pure peptides were analyzed using a HP1090 system with a 4.6 × 150 mm Phenomenox C18 (2), 5 μm 100A column eluting with solvent gradient A: B. . Where solvent A = acid in 0.1% trifluoroacetic acid in water and B = 0.09% trifluoroacetic acid. A mixture of acetonitrile: water (4: 1) was added at a flow rate of 1.0 mL / min over 20 minutes. Specific retention time, UV purity (220 nm), and solvent gradient are described for the final peptide. The hydrogen atom has been omitted from the peptide structure below for clarity.
Mass spectral analysis: Mass data based on ESI was collected using an Agilent 6200 series TOF / 6500 series Q-TOF or Thermo-LCQ Advantage system.
ppTG21 and ppTG21 derivatives ppTG21: GLFHALLHLLHSLWHLLLLHA (SEQ ID NO: 1012) (74),
Fmoc-Ala-Wang resin (5 mmol, 10 g) was placed in the peptide reactor and the resin was allowed to swell in DMF for 2 hours. The Fmoc group was then removed by the addition of a 20% (volume) solution of piperidine in DMF (150 mL), followed by stirring for 1 minute. This process was repeated 5 times. A Kaiser ninhydrin test was performed to show complete deprotection. A solution of Fmoc-His (Trt) -OH (15.0 mmol, 9.29 g) and HBTU (14.3 mmol, 5.42 g) in DMF (ca. 40 mL) at 0 ° C. with N-methylmorpholine (30 mmol, 3. 3 mL) and the mixture was maintained at 0 ° C. for 15 minutes. This solution was added to the H-Ala-Wang resin and the mixture was stirred for 1 hour at 25 ° C. and the Kaiser ninhydrin test showed that the reaction was complete. The mixture was filtered and the solid was washed with DMF (5 × 150 mL). The resulting Fmoc-His (Trt) -Ala-Wang resin product was used in subsequent steps without further processing.
After Fmoc deprotection of the peptidyl resin, the Fmoc-amino acid is sequentially coupled to the resin-bound peptide using the standard amide coupling / FMOC cleavage method described above, and H-Gly-Leu-Phe-His ( Trt) -Ala-Leu-Leu-Leu-Leu-His (Trt) -Lau-Leu-His (Trt) -Ser (tBu) -Leu-Trp (Boc) -His (Trt) -Leu-Leu-Leu- His (Trt) -Ala-Wang resin (SEQ ID NO: 1052) was supplied. The peptidyl resin was washed with MeOH (2 × 150 mL), dichloromethane (2 × 150 mL) and MeOH (2 × 150 mL). The resin was dried overnight under vacuum. A solution 0 (87.5: 5: 2.5: 2.5: 2.5, 650 mL) of TFA: thioanisole: phenol: EDT: H ₂ O was added to the peptidyl resin and the resulting suspension Was shaken for 2.5 hours and filtered. Ether (5 L) was added to the filtrate to obtain a solid. The mixture was centrifuged and the ether layer was decanted. The resulting solid was washed with ether (3x) and dried in vacuo overnight. The resulting crude was then purified by reverse phase HPLC and similar fractions were combined and then lyophilized to give 5.18 g of the desired peptide (TFA salt) as a white solid. UV purity (220 nm) = 95.4% (retention time = 9.22 min, solvent gradient A: B, 24:76 to 14:86). ESI (m / z) 2341.3430 (M + H) ^<+> .

Ac-Cys (NPys) -ppTG21: Ac-Cys (NPys) -GLFHALLHLLHSLWHLLLLHA (SEQ ID NO: 1049) (75)
Using substantially the same peptide loading and amino acid method as described above, Ac-Cys (Trt) -Gly-Leu-Phe-His (Trt) -Ala-Leu-Leu-His (Trt) -Leu-Leu -His (Trt) -Ser (tBu) -Leu-Trp (Boc) -His (Trt) -Leu-Leu-Leu-His (Trt) -Ala-Wang resin (SEQ ID NO: 1053) was provided. The peptidyl resin was washed with methanol (2 × 150 mL), dichloromethane (2 × 150 mL) and methanol (2 × 150 mL). The resin was dried overnight under vacuum. A solution of TFA: thioanisole: phenol: EDT: H ₂ O = 87.5: 5: 2.5: 2.5: 2.5, 650 mL) was added to the peptidyl resin, and the suspension was added to 2.5. Shake for hours and then filter. Ether (5 L) was added to the filtrate to obtain a solid. The mixture was centrifuged and the ether layer was decanted. The resulting solid was washed with ether (3x) and dried in vacuo overnight. The resulting crude peptide was then purified via reverse phase HPLC, similar fractions were combined, lyophilized and 800 mg (90% by UV) Ac-Cys-Gly-Leu-Phe-His- Ala-Leu-Leu-His-Leu-Leu-His-Ser-Leu-Trp-His-Leu-Leu-His-Ala-OH (SEQ ID NO: 1054) was obtained. A solution of the peptide in N, N-dimethylformamide (80 mL) was added with 2,2′-dithiodi (5-nitropyridine) (200 mg, 2 eq) and N, N-diisopropylethylamine (0.23 mL, 4 eq). Processed. The mixture was stirred for 1 hour. N, N-dimethylformamide was removed under reduced pressure to give a yellow oil (1.05 g). The resulting crude peptide was purified via reverse phase HPLC and similar fractions were combined and lyophilized to give 520 mg (66%) of the desired peptide (TFA salt) as a white solid. UV purity (220 nm) = 95.6%. Retention time = 10.80 minutes. Solvent gradient A: B = 10: 90 to 0: 100. ESI (m / z) 2640.3345 (M + H) ^<+> .

Ac-Cys (NPys) -PEG4-ppTG21: Ac-Cys (NPys) -PEG3-GLFHALLHLLHSLWHHLLLHA (SEQ ID NO: 1050) (76)
The peptide reactor was charged with Fmoc-Ala-wang resin (1.0 mmol, 2 g) and the resin was swollen in N, N-dimethylformamide for 2 hours. The Fmoc group was then cleaved by addition of a 20 volume% (volume) piperidine solution in DMF (30 mL) followed by stirring for 1 minute. This process was repeated 5 times. A Kaiser ninhydrin test was performed to show complete deprotection.

A solution of Fmoc-His (Trt) -OH (3 mmol, 1.89 g), HBTU (2.85 mmol, 1.08 g) in N, N-dimethylformamide (about 10 mL) was added to N-methylmorpholine (6 mmol, 0 .66 mL) at 0 ° C. and the mixture was maintained at 0 ° C. for 15 minutes. This solution was then added to the H-Ala-wang resin and the mixture was stirred at 25 ° C. for 1 hour, at which point the Kaiser ninhydrin test showed that the reaction was complete. The mixture was filtered and the solid was washed with N, N-dimethylformamide (5 × 30 mL). The resulting Fmoc-His (Trt) -Ala-CTC resin product was used in the next step without further processing. Following Fmoc deprotection of the peptidyl resin, Fmoc amino acids were sequentially coupled to the resin-bound peptide using the standard amide coupling / FMOC cleavage method described above. Ac-Cys (Trt) -PEG-Gly-Leu-Phe-His (Trt) -Ala-Leu-Leu-His (Trt) -Leu-Leu-His (Trt) -Ser (tBu) -Leu-Trp (Boc) ) -His (Trt) -Leu-Leu-Leu-His (Trt) -Ala-Wang resin (SEQ ID NO: 1055) was constructed and then the peptidyl resin was added to methanol (2 × 50 mL), dichloromethane (2 × 50 mL) and Wash with methanol (2 × 50 mL). The resin was dried overnight under vacuum. TFA: thioanisole: phenol: EDT: H ₂ O = 87.5: 5: 2.5: 2.5: 2.5, 150 mL) was added and the suspension was shaken for 2.5 hours. And filtered. Ether (1.2 L) was added to the filtrate to obtain a solid. The mixture was centrifuged and the ether layer was decanted. The resulting solid was washed with ether (3x) and dried in vacuo overnight. The resulting crude peptide was purified by reverse phase HPLC, the same fractions were combined, lyophilized and Ac-Cys-PEG-Gly-Leu-Phe-His-Ala-Leu-Leu-His-Leu-Leu-Leu. -His-Ser-Leu-Trp-His-Leu-Leu-Leu-His-Ala-OH (SEQ ID NO: 1056) was obtained. The crude peptide (1.10 g, purity: 85% by HPLC) in N, N-dimethylformamide (110 mL) was added to 2,2′-dithiodi (5-nitropyridine) (275 mg, 2 eq) and DIPEA (0. 32 mL, 4 eq). The mixture was stirred for 1 hour and then DMF was removed under reduced pressure to give a yellow oil. The resulting crude peptide was purified using reverse phase HPLC and similar fractions were combined and lyophilized to give 270 mg (27%) of the desired peptide (TFA salt) as a white solid. UV purity (220 nm) = 95.4%. Retention time = 9.18 minutes. Solvent gradient A: B = 14: 86 to 4:96. ESI (m / z) 2887.972 (M + H) ^<+> .

ppTG21-Cys (NPys) -OH: GLFHALLHLLHSLWHLLLLHA-Cys (NPys) -OH (SEQ ID NO: 1051) (77)
Fmoc-Cys (Trt) -CTC resin (1.0 mmol, 3.3 g) was placed in the peptide reactor. The resin was swollen in N, N-dimethylformamide for 2 hours. The Fmoc group was then cleaved with a 20% (volume) solution of piperidine in N, N-dimethylformamide (80 mL) followed by stirring for 1 minute. This process was repeated 5 times. A Kaiser ninhydrin test was performed to show complete deprotection. A solution of Fmoc-Ala-OH (3 mmol, 0.93 g), HBTU (2.85 mmol, 1.08 g) in N, N-dimethylformamide (30 mL) was added at 0 ° C. with N-methylmorpholine (6 mmol, 0.003 g). 66 mL) The mixture was maintained at 0 ° C. for 15 minutes. This solution was then added to the H-Cys (Trt) -CTC resin and the mixture was stirred at 25 ° C. for 1 hour, at which point the Kaiser ninhydrin test showed that the reaction was complete. The mixture was filtered and the solid was washed with N, N-dimethylformamide (5 × 30 mL). This Fmoc-Ala-Cys (Trt) -CTC resin was used in the next step without further treatment.

The following amino acids were sequentially coupled to the resin bound peptide using the standard amide coupling / FMOC cleavage method described above. H-Gly-Leu-Phe-His (Trt) -Ala-Leu-Leu-His (Trt) -Leu-Leu-His (Trt) -Ser (tBu) -Leu-Trp (Boc) -His (Trt)- Leu-Leu-Leu-His (Trt) -Ala-Cys (Trt) -CTC resin (SEQ ID NO: 1057) was constructed. The peptidyl resin was washed with methanol (2 × 100 mL), dichloromethane (2 × 100 mL) and methanol (2 × 100 mL). The resin was dried overnight under vacuum. TFA: thioanisole: phenol: EDT: H ₂ 0 = 87.5: 5: 2.5: 2.5: 2.5, 150 mL of solution was added and the suspension was shaken for 2.5 hours. And then filtered. Ether (1.2 L) was added to the filtrate to give a solid. The mixture was centrifuged and the ether layer was decanted. The crude peptide was washed with ether (3x) and dried in vacuo overnight. The resulting residue was purified by reverse phase HPLC, the same fractions were combined, lyophilized and H-Gly-Leu-Phe-His-Ala-Leu-Leu-His-Leu-Leu-His-Ser- Leu-Trp-His-Leu-Leu-Leu-His-Ala-Cys-OH (SEQ ID NO: 1058) was obtained. 1.4 g (HPLC purity 75%) of this peptide was dissolved in N, N-dimethylformamide (140 mL), 2,2′-dithiodi (5-nitropyridine) (217 mg, 2 eq) and N, N-diisopropylethylamine (0.25 mL, 4 eq) was added. The mixture was stirred for 1 hour and then N, N-dimethylformamide was removed under reduced pressure to give a yellow oil. The resulting crude peptide was purified by reverse phase HPLC and similar fractions were combined and lyophilized to give 280 mg (25%) of the desired peptide (TFA salt) as a white solid. UV purity (220 nm) = 95.3%. Retention time = 9.88 minutes. Solvent gradient A: B = 11: 89 to 1:99. ESI (m / z) 1299.9 (M / 2 + H) ⁺ , 866.7 (M / 3 + H) ⁺ .

Ac-Cys-ppTG21: Ac-CGLFHALLHLLHSLWHLLLLHA (SEQ ID NO: 1059) (78),
This peptide was synthesized using a solid phase peptide synthesis (SPPS) procedure similar to that described in the previous examples. UV purity (220 nm) = 95.6%. Retention time = 10.25 minutes. Solvent gradient A: B = 14: 86 to 4:96. ESI (m / z) 24866.3333 (M + H) ^<+> .

Ac-Cys-dPEG4-ppTG21: Ac-Cys-dPEG4-GLFHALLHLLHSLWHLLLLHA (SEQ ID NO: 1060) (79),
This peptide was synthesized using a solid phase peptide synthesis (SPPS) procedure similar to that described in the previous examples. UV purity (220 nm) = 95.3%. Retention time = 8.61 minutes. Solvent gradient A: B = 18: 82 to 8:92. ESI (m / z) 2733.5157 (M + H) ^<+> .

ppTG21-Cys: GLFHALLHLLHSLWHLLLLHAC (SEQ ID NO: 1061) (80),
This peptide was synthesized using a solid phase peptide synthesis (SPPS) procedure similar to that described in the previous examples. UV purity (220 nm) = 95.3%. Retention time = 10.26 minutes. Solvent gradient A: B = 21: 79 to 11:89. ESI (m / z) 24444.3673 (M + H) ^<+> .

Ac-Gly (propargyl) -ppTG21: Ac-Gly (propargyl) -GLFHALLHLLHSLWHLLLLHA (SEQ ID NO: 1062) (81
This peptide was synthesized using a solid phase peptide synthesis (SPPS) procedure similar to that described in the previous examples. UV purity (220 nm) = 95.4%. Retention time = 10.56 minutes. Solvent gradient A: B = 14: 86 to 4:96. ESI (m / z) 24788.4154 (M + H) ^<+> .

SPDP-dPEG8-ppTG21: SPDP-dPEG8-GLFHALLHLLHSLWHLLLLHA (SEQ ID NO: 1063) (82)
To a solution of ppTG21 (74) peptide hexatrifluoroacetate (7.7 mg, 0.0025 mmol) in anhydrous N, N-dimethylformamide (1.0 mL) was added {27-[(2,5-dioxopyrrolidine. -1-yl)) oxy] -27-oxo-3,6,9,12,15,18,21,24-octaoxaheptakos-1-yl} -3- (pyridin-2-yldisulfanyl) propane Amide
A solution of (FCD13185003, 1.97 mg, 0.0027 mmol) was added followed by N, N-diisopropylethylamine (7.0 μM) at room temperature. The reaction was stirred at room temperature for 18 hours and concentrated under reduced pressure using Genevac. The crude material was purified using reverse phase chromatography using the following conditions to give the title compound as a glassy solid (3.3 mg, 40%).

Purification conditions The residue was dissolved in dimethyl sulfoxide (1 mL) and purified by reverse phase HPLC. Column: Waters Sunfire C18, 19 × 100, 5u. Mobile phase A: 0.05% TFA in water (v / v). Mobile phase B: 0.05% TFA in acetonitrile (v / v). Gradient: 10.5 minutes 60.0% H ₂ O / 40.0% acetonitrile to 30.0% H ₂ O / 70.0% acetonitrile with a linear, 30.0% H ₂ O at 10.5 minutes From 70.0% acetonitrile to 0% H ₂ O / 100% acetonitrile in a straight line from 11.0 minutes to 12.0 minutes to 0% H ₂ O / 100% acetonitrile. Flow rate: 25 mL / min.

QC conditions Column: Waters Atlantis dC18, 4.6 × 50, 5u. Mobile phase A: 0.05% TFA in water (v / v). Mobile phase B: 0.05% TFA in acetonitrile (v / v). Gradient: 75.0% H ₂ O / 25.0% in 4.0 minutes to 5% H ₂ O / 95% acetonitrile in a straight line, 4.0% H ₂ O / 95% acetonitrile up to 5.0 minutes. Maintenance. Flow rate: 2 mL / min. Retention time = 3.33 minutes. Observed mass M / Z = 741.65. Method E: ESIM / Z = 2963.0

KALA and KALA derivatives KALA: WEAKLAKALAKALAKHLAKALAKALKACEA (SEQ ID NO: 869) (83)
This peptide was synthesized using a solid phase peptide synthesis (SPPS) procedure similar to that described in the previous examples. UV purity (220 nm) = 95.6%. Retention time = 10.03 minutes. Solvent gradient A: B = 54: 46 to 44:56. ESI (m / z) 310.8272 (M + H) ^<+> .

Cys-KALA: CWEAKLAKALAKALAKHLAKALAKALKAACEA (SEQ ID NO: 1064) (84)
This peptide was synthesized using a solid phase peptide synthesis (SPPS) procedure similar to that described in the previous examples. UV purity (220 nm) = 97.2%. Retention time = 8.05 minutes. Using a solvent gradient of water (0.1% THF: acetonitrile / water (4: 1, 0.1% TFA), Phenomenex Kinexex (100 × 4.6 mm), flow rate = 1 mL / min, 10 minutes, ESI ( m / z) 1618.6 (M + 2H) ²⁺ , 1079.3 (M + 3H) ³⁺ , 809.8 (M + 4H) ⁴⁺ .

Serine for cysteine (KALA) -Cys (NPys) -OH,
WEAKLAKALAKALAKHLAKALAKALKASEA-Cys (NPys) -OH (SEQ ID NO: 1065) (85)
This peptide was synthesized using a solid phase peptide synthesis (SPPS) procedure similar to that described in the previous examples. UV purity (220 nm) = 95.8%. Retention time = 9.48 minutes, solvent gradient A: B (50:50 to 40:60). ESI (m / z) 3372.88 (M + H) ⁺

Ac-Cys (NPys) -Serine (KALA) -OH, Ac-Cys (NPys) -WEAKLAKALAKALAKHLAKALAKALKASEA (SEQ ID NO: 1066) (86)
This peptide was synthesized using a solid phase peptide synthesis (SPPS) procedure similar to that described in the previous examples. UV purity (220 nm) = 95.7%. Retention time = 9.78 minutes, solvent gradient A: B = 46: 54 to 36:64. ESI (m / z) 3414.9079 (M + H) ^<+> .

Ac-Cys (NPys) -PEG- (KALA) serine substitution: Ac-Cys (NPys) -PEG-WEAKLAKALAKAKLAKLAKALKALASEA (SEQ ID NO: 1067) (87)
This peptide was synthesized using a solid phase peptide synthesis (SPPS) procedure similar to that described in the previous examples. UV purity (220 nm) = 95.1%. Retention time = 9.23 minutes, solvent gradient A: B = 53: 47 to 37:63. ESI (m / z) 3662.0715 (M + H) ⁺ .

Preparation of Cas9 constructs and guide RNAs Expression and purification of Cas9 constructs and guide RNAs have been described in the literature (Jinek et al., A programmable dual-RNA-guided DNA endocrinese in adaptive bacterial immunity. 21 (2012)). Briefly, S.I. The E. coli codon optimized gene encoding the pyogenes Cas9 M1C / C80S protein (SEQ ID NO: 850), hexa-histidine (SEQ ID NO: 1068), maltose binding protein tag at the N-terminus, and two nucleic acid localization signals at the C-terminus (NLS) (each NLS is PKKRKV (SEQ ID NO: 830)) or three NLS (each NLS is PKKRKV (SEQ ID NO: 830) and mCherry (SEQ ID NO: 915) as a fusion protein and inserted into the bacterial protein expression plasmid. The amino acid sequence of the Cas9 construct set forth at number 1013 is called Y1C80S-2N, and the amino acid sequence of the Cas9 construct set forth at SEQ ID NO: 1015 is called the Cas9 construct Y1C80S-3N-m.

The plasmid was transformed into E. coli Rosetta (DE3). Bacteria were inoculated in Lysogeny broth medium at an optical density of ₆₀₀ nm (OD ₆₀₀ nm) of 0.05 and grown at 37 ° C. and 170 rpm. Expression was induced by addition of 0.2 mM isopropyl-β-D-thiogalactopyranoside (IPTG) at an OD value of 0.8 μm and grown at 16 ° C. for 16 hours. Bacteria were inoculated into lysogen broth (LB) medium at an optical density of 600 (OD _{600 nm} ) of 0.05 and grown at 37 ° C. and 170 rpm. Pellet bacteria, discard supernatant, then 20 mM 4- (2-hydroxyethyl) -1-piperazineethanesulfonic acid (HEPES), 500 mM potassium chloride (KCl), 5 mM tris (2-carboxyethyl) phosphine (TCEP) Dissolved by sonication in 10 mM imidazole, pH 7.5. Clarified lysate was captured on Ni-NTA agarose (Qiagen) and washed thoroughly with 20 mM HEPES, 500 mM KCl, 5 mM TCEP, 10 mM imidazole, pH 7.5, 20 mM HEPES, 250 mM KCl, 5 mM TCEP, 300 mM imidazole, 10% glycerol, pH 7.5, eluting at 4 ° C. Remove 6 × His-MBP tag (“6 × His” disclosed as SEQ ID NO: 1068) overnight with tobacco etch virus (TEV) protease, while 20 mM HEPES, 300 mM KCl, 5 mM TCEP, 10 Dialyzed in% glycerol, pH 7.5. The Cas9 construct was separated from the tag by capture on a heparin SP column (GE Healthcare) and eluting linearly with 300 mM to 1 M KCl. Finally, the Cas9 construct was further purified on a Superdex S200 HiLoad column (GE Healthcare) in 20 mM HEPES, 150 mM KCl, 10% glycerol, pH 7.5. At this point, the Cas9 construct was concentrated to a concentration determined by UV absorbance of about 15-20 mg / mL, purity was assessed by SDS-PAGE, and aliquots were snap frozen in liquid nitrogen. Aliquots were stored at -80 ° C.

  FIG. 3A represents the mass spectrum of the intact Cas9 construct Y1C80S-3N-m.

DNA encoding the T7 promoter and guide RNA template were transcribed in vitro using a T7 polymerase and ribonucleotide solution mixture at 8 mM and pH 7.5 overnight at 37 ° C. The DNA template was digested with DNAse for 1 hour at 37 ° C. and RNA was purified on a denaturing PAGE gel. The RNA band was excised from the PAGE gel, eluted with 300 mM sodium acetate (NaOAc) pH 5.0 and precipitated with ethanol. The RNA pellet was resuspended in 20 mM HEPES, 150 mM KCl, 10% glycerol, pH 7.5. The guide RNA is refolded by incubating at 70 ° C. for 5 minutes, cooled to room temperature, magnesium chloride (MgCl ₂ ) is added to a final concentration of 1 mM, incubated at 50 ° C. for 5 minutes, and finally to room temperature Cooled down. The concentration was determined by UV absorbance and stored at -80 ° C.

Preparation of Cas9-ASGPR ligand conjugate Example Y53aASGPRL: ASGPR ligand (Compound 53, N- (1,3-bis [(1- {1-[(1S, 2R, 3R, 4R, 5S) -4- (acetyl Amino) -2,3-dihydroxy-6,8-dioxabicyclo [3.2.1] oct-1-yl] -2,5,8,11-tetraoxatridecan-13-yl} -1H- 1,2,3-triazol-4-yl) methoxy] -2-{[(1- {1-[(1S, 2R, 3R, 4R, 5S) -4- (acetylamino) -2,3-dihydroxy -6,8-dioxabicyclo [3.2.1] oct-1-yl] -2,5,8,11-tetraoxatridecan-13-yl} -1H-1,2,3-triazole- 4-yl) methoxy] methyl} Lopan-2-yl) -3,31-dioxo-1- (pyridin-2-yldisulfanyl) -7,10,13,16,19,22,25,28-octoxa-4,32-diazaoctatria Contan 38-amide) was reconstituted to 8 mM in dimethyl sulfoxide (DMSO) to give the ligand fragment 53a (shown below). The ligand fragment 53a was then diluted 10-fold in 20: 1 HEPES, 150 mM KCl, 10% glycerol, pH 7.5, and the Cas9 construct (Y1C80S-3N- added directly to m) and incubated at room temperature for 1-2 hours. The labeled Cas9 construct, Y53aASGPRL, was desalted using a Zeba Spin column (Thermo Fisher).

  Using a Agilent 1200 liquid chromatograph (LC) equipped with a reverse phase C4 column (150 mm x 1.0 mm, Restek), labeling efficiency was determined by mass spectral analysis on the intact Cas9 construct, Y53aASGPRL, and electrospray ionization Measured using a Thermo LTQ-Orbitrap-XL mass spectrometer equipped with an (ESI) source. Raw mass spectra were observed using Xcalibur software (version 2.0.7, Thermo) and mass spectral deconvolution integration was performed using ProMass software (version 2.5SR-1, Novatia).

FIG. 3B represents the mass analysis of Cas9 construct Y53aASGPRL, which is Cas9 construct Y53aASGPRL2 labeled with two copies of fragment 53a, one at each of cysteines at positions 1 and 574. Labeled through formation of a disulfide bond with the S atom (addition of 2 × 2165 Da).
Y1C80S-3N-m (1) and Y53aASGPRL (2) were analyzed using 20 mM HEPES, 150 mM KCl, 10% glycerol, pH 7.5, analytical size exclusion chromatography (Superdex S200 10 / 30GL), 50 μM. Run with 50 μL. Elution volume for (1): 11.56 mL; and elution volume for (2): 11.44 mL.

RNP assembly and gene editing assay Example Y53a-C574-ASGPRL-RNP-EMX1: (53) RNP (S. (SEQ ID NO: 1026)
29.5 μL of a solution (15.6 μL, 222 μｐ, 3463 pmol) of EMXl · sgRNA guide sequence (SEQ ID NO: 907) in 1 mM MgCl ₂ , 20 mM HEPES, 150 mM KCl, 10% glycerol (pH 7.5). 1 mM MgCl ₂ , 20 mM HEPES, 150 mM KCl, 10% glycerol (pH 7.5) buffer was added to obtain an EMX1 · sgRNA guide sequence (SEQ ID NO: 907) in 45 μl, 76.8 μM.

S. pyogenesCas9-mutated C80S (amino acid sequence) -3NLS labeled with compound (53) (12 μL, 80 μM, 960 pmol) in 20 mM HEPES, 150 mM KCl, 10% glycerol (pH 7.5) buffer mCherry (SEQ ID NO: 1026) [referred to herein as Y53a-C574-ASGPRL and labeled with a compound (53) that is good for the same method as described for Cas9 construct Y53aASGPRL] Add 3 μL of MgCl ₂ to 150 mM KCl, 10% glycerol (pH 7.5) buffer and add Cas9 in 1 mM MgCl ₂ , 20 mM HEPES, 150 mM KCl, 10% glycerol (pH 7.5) buffer. Construct Y53a-C To obtain a 74-ASGPRL15μl.

  Transfer 15 μL of the above EMX1 · sgRNA guide sequence (SEQ ID NO: 907) solution (15 μL, 76.8 μM, 1152 pmol) to another 2 mL Eppendorf tube, and add Y53a-C574-ASGPRL (15 μL, 64 μM, 960 pmol) to this, Pipetting 5 times and mixing gave Y53a-C574-ASGPRL • RNP construct (30 μM, 32 μM). This RNP construct is referred to herein as Y53a-C574-ASGPRL-RNP-EMX1. The reaction mixture was incubated at 37 ° C. (water bath) for 10 minutes and then used directly in biological or fluorescence microscopy experiments.

Example Y53a-C574-ASGPRL-RNP-EMX1: purulent Cas-mutant M1C && C80S (amino acid sequence) -compound (53RNP) (PCSK9 single guide sgRNA sequence 1 (SEQ ID NO: 896)) 3NLS and mCherry (SEQ ID NO: 1015)
A solution of PCSK9 single guide sgRNA sequence (SEQ ID NO: 896) in 1 mM MgCl ₂ , 20 mM HEPES, 150 mM KCl, 10% glycerol (pH 7.5) buffer, 0.96 μL of 1 mM in 20 mM HEPES. MgCl ₂ , 20 mM HEPES, 150 mM KCl, 10% glycerol (pH 7.5) buffer was added to obtain 5 μL of PCSK9 single guide sgRNA sequence 1 (SEQ ID NO: 896) at 64 μM.

Cas9 construct Y1C80S-3N-m (4 μL, 80 μM, 320 pmol) in 20 mM HEPES, 150 mM KCl, 10% glycerol (pH 7.5) buffer was added to 20 mM HEPES, 150 mM KCl, 10% glycerol (pH 7.5). ) Add 1 μL of 5 mM MgCl ₂ in buffer and add Cas9 construct Y53a-C574-ASGPRL (64 μM in 1 mM MgCl ₂ , 20 mM HEPES, 150 mM KCl, 10% glycerol (pH 7.5) buffer) 5 μL) was obtained.

The above Y53aASGPRL solution (5 μL, 64 μM, 320 pmol) was added to the solution of PCSK9 single guide sgRNA sequence 1 (SEQ ID NO: 896) (5 μL, 76.8 μM, 384 pmol), mixed by pipetting 5 times, and Y53aASGPRL Obtained the RNP construct (10 μL at 32 μM), which is referred to herein as the RNP construct Y53aASGPRL-RNP-PCS1. The reaction mixture was incubated at 37 ° C. (water bath) for 10 minutes and then used directly in biological or fluorescent microscopy experiments.
Using the same procedure, the following RNPs were prepared:
Example Y53aASGPRL-RNP-EMX1: The Cas9 construct Y53aASGPRL and the EMX1 guide RNA sequence (SEQ ID NO: 907) were used.
Example Y53aASGPRL-RNP-PCS4: Cas9 construct Y53aASGPRL and the PCSK9 guide RNA sequence (SEQ ID NO: 906) were used.

General protocol:
Gene editing in human hepatocytes was performed by T7 endonuclease 1 (T7E1) endonuclease assay. Briefly, 80000 HepG2 (ASGPR positive-ATCCnb.HB-8065) or SkHep (ASGRP negative-ATCCnb.HTB-52) cells were seeded in 24-well plates. Assemble the Cas9 guide RNA ribonucleoprotein (Cas9 • RNP) by incubating the following Cas9 constructs: Y1C80S-3N-m and Y53aASGPRL with a 1: 1.2 molar ratio of guide RNA for 10 minutes at 37 ° C. It was.

Cas9 • RNP +/− ppTG21 was dispensed directly into the culture medium and incubated at 37 ° C., 5% CO ₂ in a humidified atmosphere, thereby allowing the cells to reach 50 pmol in the presence or absence of the ppTG21 endosomal degradation agent. Treated with Cas9 • RNP. After 48 hours of incubation, the culture medium was removed and the cells were lysed with QuickExtract buffer (Epibio) for 5 minutes at room temperature. The supernatant was transferred to a tube and the sample was heated at 65 ° C. for 20 minutes, followed by heating at 95 ° C. for 20 minutes. Genomic DNA concentration was measured by UV absorbance. Specific loci (following sequences) were used to amplify the locus of interest and the polymerase chain reaction (PCR) product was visualized on an agarose gel and quantified by comparison with a standard. 200 ng of PCR product was thawed and rehybridized and digested with T7E1 endonuclease at 37 ° C. for 30 minutes. Editing efficiency was determined by quantification of the cleaved PCR product.

EMX1 locus primer:
Forward direction: 5′-GCCATCCCCCTCTGTGAATGTTAGAC-3 ′ (SEQ ID NO: 1017)
Reverse direction: 5′-GGAGATTGGAGACACGAGAGACAG-3 ′ (SEQ ID NO: 1018)

PCSK9 exon 1 locus primer:
Forward direction: 5′-CCAGCTCCCCAGCCAGGATC-3 ′ (SEQ ID NO: 1019)
Reverse direction: 5′-ATCGTGCCAAGCGAAGAG-3 ′ (SEQ ID NO: 1020)

PCSK9 exon 4 & 5 locus primers:
Forward direction: 5'-TGATGGCCCTTGGACAGTTACCC-3 '(SEQ ID NO: 1021)
Reverse direction: 5′-GGTCCAGATGGAGAGAGACCA-3 ′ (SEQ ID NO: 1022)

  HepG2 and SkHep cells were obtained using 50 pmol of RNP construct Y53aASGPRL-RNP-EMX1Y1C80S-3N-mNPRN-EMX1 and increasing concentrations of ppTG21 endosomal lytic peptides (62.5, 250, 1000 and 2000 nM). Processed in the presence (co-incubation). RNP construct only treatment (no endosomal lytic peptide) and RNP construct lipofection treatment were used as negative and positive controls, respectively. Cells were treated for 48 hours before editing was assessed by the T7E1 endonuclease assay. The editing efficiency is noted in FIG.

  HepG2 and SkHep cells were treated with 50 μL of Cas9RNPY53aASGPRL-RNP-PCS4 in the presence of increasing concentrations of ppTG21 endosomal lytic peptides (250 and 1000 nM). Cells were treated for 48 hours before editing was assessed by the T7E1 endonuclease assay. The editing efficiency is noted in FIG.

ASGPR-Cas9 Microscopic Imaging Test Materials / Reagents:
SKHep cells (ATCC HTB-52)
HepG2 cells (ATCC HB-8065)
Growth medium: DMEM high glucose (ThermoFisher 1965-092)
10% fetal bovine serum, heat-inactivated (ThermoFisher 16000-044)
1 mM non-essential amino acid (ThermoFisher 11140-050)
2 mM L-glutamine (ThermoFisher 25030-081)
100 units / mL penicillin / streptomycin (ThermoFisher 10378-016)
MatTek glass bottom well (MatTek Corp. P35G-1.5-14-C)
Dextran 647- (Life Technologies D34682)
Hoechst 3328- (Life Technologies 62249) used at -1 μg / mL Collagen- (Life Technologies A10483-1) -5 ug / cm ² used Y53aASGPRL-RNP-EMX1 and Y1C80S-3N-MXRN-P used. An image is shown in FIG.
protocol:
-Plate collagen-coated MatTek wells at 40,000 / well in growth medium-Change to fresh medium after 24 hours-Treat with Dextran 647 (500 ng / mL) for 4 hours.
-4 hours later, Hoechst is added to the medium for 5 minutes.
-Change to fresh medium.
Remove the medium and add Y1 • C80S-3N-m-RNP-EMX1 • RNP construct 64 μM or Y53 • ASGPRL-RNP-EMX1 • RNP construct at 64 μM.
Image with a Zeiss rotating disk microscope for 1 hour in live cell conditions and collect images every 5 minutes.
Images were processed with Zen software (Carl Zeiss, Inc).

Pharmacological Data The practice of the invention to treat diseases modulated by targeting the asialoglycoprotein receptor (ASGPR) with the compounds of the invention is an activity in one or more functional assays described below. Can be proved by. Sources are listed in parentheses.

ASPRr ligand SPR binding measurement:
All SPR measurements using the compounds were performed at 25 ° C. using a Biacore 3000 (GE Healthcare). Biotinylated ASGPR typically uses a custom sensor chip with Neutravidin (Pierce Biochemical) immobilized by standard amine coupling to an SA sensor chip (GE Healthcare) or CM5 sensor chip (GE Healthcare). Was immobilized at 2000 to 3000 resonance units (Ru). Running buffer is HBS (10 mM HEPES, 150 mM NaCl), 20 mM CaCl ₂ , 0.01% p20, 3% DMSO, or 50 mM Tris, 150 mM NaCl, 50 mM CaCl ₂ , 0.01% P20, 3% DMSO, pH 7.5. The compound was diluted in running buffer at a concentration of 900 μM and serially diluted 3-fold to 3.3 μM. Compound solution was injected at 50 uL / min for 1 minute followed by dissociation for 2 minutes for each concentration for 1 minute. For multimeric conjugates (dimers, trimers), conjugates were serially diluted to a concentration of 100 nM or 10 nM in running buffer. The conjugate was injected for 2 minutes and the off rate was detected for 300 or 600 seconds. After completion of the off-phase data, 900 μM GalNAc injection was used to displace the compound and return the receptor surface to the free state. All data were processed using a scrubber 2 (Biologic Software, Inc.), zeroed, aligned for excluded volume effects, referenced and corrected. The K _D S was determined by fitting the steady state binding response of the compound and single conjugated molecule in scrubber 2. Was treated with a scrubber 2 the K _D of the multimeric conjugate showing the kinetics response and adapted to BiaEval (GE Healthcare), it was extracted on and off speed parameter to calculate the K _D. The value reflects the standard deviation of multiple experiments.
The following results are obtained for the SPR binding assay, where each assay is reported separately or the run number (n) and standard deviation are recorded.

SPR binding measurement of RNP construct:
SPR: All experiments were performed on a Biacore 3000 (GE Healthcare) using a commercially available streptavidin sensor (Sensor Chip SA, GE Healthcare) at 25 ° C.
The level of ASGPr / HI / CRD protein immobilization depends on the experiment and is described in the individual experimental protocols. All experiments were performed at a flow rate of 50 uL / min. In all experiments, the streptavidin sensor surface was used as a reference. All obtained data were processed using Scrubber2 software (BioLogic Software), zeroed, x-aligned and corrected for reference and baseline. The curves were fitted using a 1: 1 kinetic binding model in a scrubber.

reagent:
ASGPr HI CRD: For capture on the streptavidin surface, ASGPr HI CRD was derivatized with maleimide-PEG2-biotin (Pierce) reacted with an isolated free cysteine as described above. It was. For protein conjugates, the surface density was kept low to reduce the congestion on the surface that caused steric collisions.

Reagents: The ASGPr-biotin RNP construct described above is supplied as a 32 μM stock in 20 mM HEPES pH 7.5, 150 mM KCl, 20 mM CaCl ₂ , 5 mM MgCl ₂ , 0.01% p20. This, along with RNP, served as a running buffer for SPR. For Cas9-ribonucleoprotein, this stock was diluted 10-fold with running buffer and then re-diluted to a maximum concentration of 10 nM. ASGPr was immobilized on a streptavidin sensor at 50 and 200 Ru. A single injection at a concentration of 10 nM showed a clear binding that could be competed primarily by injection of excess (900 μM) N-acetyl-galactosamine (GalNAc), similar to that observed with small molecule complexes. A control RNP construct without the ASGPr ligand (Y1C80S-3N-m-RNP-EMX1) was also injected at the highest concentration (10 nM) but did not show any binding to the immobilized receptor. Proteins were serially diluted from 10 nM to 2-fold in running buffer to obtain affinity at both densities. Between each concentration, 900 μM GalNAc was injected to remove RNP from the receptor. Responses from a range of concentrations were processed using the scrubber 2 described above and fitted to a 1: 1 binding model.

  Throughout this application, various publications are referenced. The entire disclosure of these publications is incorporated herein by reference for all purposes.

  It will be apparent to those skilled in the art that various modifications and variations can be made in the present invention without departing from the scope or spirit of the invention. Other embodiments of the invention will be apparent to those skilled in the art from consideration of the specification and practice of the invention disclosed herein. It is intended that the specification, including the examples, be considered as exemplary only, with a true scope and spirit of the invention being indicated by the appended claims.

The following are exemplary embodiments:
1. A compound of formula (A-1), (A-2), or (A-3), or a pharmaceutically acceptable salt thereof:
Where
R ^xx -H, -alkyl, -cycloalkyl, -alkenyl, alkynyl, -aryl, -heteroaryl, -OR ^Five , -N (R ^Four -R ^Five , -SR ^Five And R ^xx -CH ₂ -Groups are each independently -O-, -S-, -N (R ^Four )-May be replaced with a heteroatom group selected from ^xx -CH _Three Is -N (R ^Four ) ₂ , -OR ^Four , And -S (R ^Four Wherein the heteroatom group is separated by at least two carbon atoms, and the alkyl, alkenyl, alkynyl, and cycloalkyl each have one or more halo atoms May be replaced with
R ^yy Are -CN, -CH ₂ -CN, -C≡CH, -CH ₂ -N _Three , -CH ₂ -NH ₂ , -CH2-N (R ^Four ) -S (O) ₂ -R ^Five , -CH ₂ -CO ₂ H, -CO ₂ H, -CH ₂ -OH, -CH ₂ -SH, -CH-CH-R ^Five , -CH ₂ -R ^Five , -CH ₂ -S-R ^Five , -CH ₂ -N (R ^Four -R ^Five , -CH ₂ -N (R ^Four ) -C (O) -R ^Five , -CH ₂ -N (R ^Four ) -C (O) -O-R ^Five , -CH ₂ -N (R ^Four ) -C (O) -N (R ^Four -R ^Five , -CH ₂ -O-R ^Five , -CH ₂ -O-C (O) -R ^Five , -CH ₂ -O-C (O) -N (R ^Four -R ^Five , -CH ₂ -O-C (O) -O-R ^Five , -CH ₂ -S (O) -R ^Five , -CH ₂ -S (O) ₂ -R ^Five , -CH ₂ -S (O) ₂ -N (R ^Four -R ^Five , -C (O) -NH ₂ , -C (O) -O-R ^Five , -C (O) -N (R ^Four -R ^Five Or aryl or heteroaryl, wherein said aryl or heteroaryl is optionally R ^Five Is replaced with
Each R ¹ Are independently -CN, -CH ₂ -CN, -C≡CH, -CH ₂ -N _Three , -CH ₂ -NH ₂ , -CH ₂ -N (R ^Four ) -S (O) ₂ -R ^Five , -CH ₂ -CO ₂ H, -CO ₂ H, -CH ₂ -OH, -CH ₂ -SH, -CH-CH-R ^Five , -CH ₂ -R ^Five , -CH ₂ -S-R ^Five , -CH ₂ -N (R ^Four -R ^Five , -CH ₂ -N (R ^Four ) -C (O) -R ^Five , -CH ₂ -N (R ^Four ) -C (O) -O-R ^Five , -CH ₂ -N (R ^Four ) -C (O) -N (R ^Four -R ^Five , -CH ₂ -O-R ^Five , -CH ₂ -O-C (O) -R ^Five , -CH ₂ -O-C (O) -N (R ^Four -R ^Five , -CH ₂ -O-C (O) -O-R ^Five , -CH ₂ -S (O) -R ^Five , -CH ₂ -S (O) ₂ -R ^Five , -CH ₂ -S (O) ₂ -N (R ^Four -R ^Five , -C (O) -NH ₂ , -C (O) -O-R ^Five , -C (O) -N (R ^Four -R ^Five Or aryl or heteroaryl, wherein the aryl or heteroaryl is optionally R ^Five Is replaced with
Or R ¹ -Z-X-Y, -Z-Y, -X-Y, -Z-X ⁺ Y ^- , -X ⁺ Y ^- , -Z-X ^- Y ⁺ , -X ^- Y ⁺ Or -Y,
X is a linker;
X ⁺ Is a positively charged linker,
X ^- Is a negatively charged linker,
Whether Y is a ribonucleoprotein or endonuclease containing a site-specific modified polypeptide, Y is a site-specific modified polypeptide, Y is a Cas9 ribonucleoprotein, or Y is a Cas9 protein , Y is a single guide RNA sequence (sgRNA) or Y is a dual guide RNA sequence comprising CRISPR RNA (crRNA) and transactivated crRNA (tracrRNA);
Y ⁺ Is a positively charged ribonucleoprotein or endonuclease containing a site-specific modified polypeptide, or Y ⁺ Is a positively charged site-specific modified polypeptide or Y ⁺ Is a positively charged Cas9 protein,
Y ^- Is a negatively charged ribonucleoprotein or endonuclease containing a site-specific modified polypeptide, or Y ^- Is a negatively charged site-specific modified polypeptide or Y ^- Is a negatively charged Cas9 ribonucleoprotein or Y ^- Is a negatively charged sgRNA or Y ^- Is a negatively charged dual guide RNA sequence comprising CRISPR RNA (crRNA) and transactivated crRNA (tracrRNA);
Z is absent or —C≡C—, —CH═CH—, —CH ₂ -, -CH ₂ -O-, -C (O) -N (R ^Four )-, -CH ₂ -S-, -CH ₂ -S (O)-, -CH ₂ -S (O) ₂ -, -CH ₂ -S (O) ₂ -N (R ^Four )-, -C (O) -O-, -CH ₂ -N (R ^Four )-, -CH ₂ -N (R ^Four ) -C (O)-, -CH ₂ -N (R ^Four ) -S (O) ₂ -, -CH ₂ -N (R ^Four ) -C (O) -O-, -CH ₂ -N (R ^Four ) -C (O) -N (R ^Four )-, -CH ₂ -O-C (O)-, -CH ₂ -O-C (O) -N (R ^Four )-, -CH ₂ -O-C (O) -O-, or aryl or heteroaryl, wherein the aryl or heteroaryl is optionally R ^Five Is replaced with
R ² Are —OH, —N _Three , -N (R ^Three ) ₂ , -N (R ^Three ) -C (O) -R ^Three , -N (R ^Three ) -C (O) -N (R ^Three ) ₂ , -N (R ^Three ) -C (O) -OR ^Three , -N (R ^Three ) -S (O) ₂ -R ^Three , Tetrazole, or triazole, wherein the tetrazole and triazole are optionally R ^Three Is replaced with
Each R ^Three Are independently -H,-(C ₁ -C _Five ) Alkyl, halo-substituted (C ₁ -C _Five ) Alkyl, halo-substituted (C _Three -C ₆ ) Cycloalkyl,-(C ₁ -C _Five ) Alkenyl,-(C ₁ -C _Five ) Alkynyl, halo-substituted- (C ₁ -C _Five ) Alkenyl, halo-substituted- (C ₁ -C _Five ) Alkynyl or (C _Three -C ₆ ) Cycloalkyl, the alkyl or cycloalkyl —CH ₂ -Groups are each independently -O-, -S-, and -N (R ^Four )-May be replaced with a heteroatom group selected from: -CH _Three Each independently represents —N (R ^Four ) ₂ , -OR ^Four , And -S (R ^Four A heteroatom group selected from: wherein the heteroatom group is separated by at least two carbon atoms;
Each R ^Four Are independently -H,-(C ₁ -C ₂₀ ) Alkyl,-(C ₁ -C ₂₀ ) Alkenyl,-(C ₁ -C ₂₀ ) Alkynyl or (C _Three -C ₆ ) 1-6 —CH of cycloalkyl, alkyl or cycloalkyl separated by said at least two carbon atoms ₂ Each group independently represents —O—, —S—, or —N (R ^Four )-May be replaced with a heteroatom independently selected from -CH _Three Are each independently -N (R ^Four ) ₂ , -OR ^Four , And -S (R ^Four ), Wherein the heteroatom group is separated by at least two carbon atoms and the alkyl, alkenyl, alkynyl, and cycloalkyl may be substituted with a halo atom Often,
Each R ^Five Are independently -H, (C _Three -C ₂₀ ) Cycloalkyl,-(C ₁ -C ₆₀ ) Alkenyl,-(C ₁ -C ₆₀ ) Alkynyl or (C ₁ -C ₆₀ ) Alkyl, 1-6 —CH of said cycloalkyl ₂ A group, or 1-20 -CH of said alkyl ₂ -Groups are each independently -O-, -S-, and -N (R ^Four )-May be exchanged with a heteroatom independently selected from, wherein the heteroatom is separated by at least two carbon atoms and the alkyl —CH _Three Are each independently -N (R ^Four ) ₂ , -OR ^Four , And -S (R ^Four And the heteroatom group is separated by at least two carbon atoms; and the alkyl, alkenyl, alkynyl, and cycloalkyl may be substituted with a halo atom. Good.
2. A compound of formula (B), or a pharmaceutically acceptable salt thereof:
Where
R ¹ -Z-X-Y, -Z-Y, -X-Y, -Z-X ⁺ Y ^- , -X ⁺ Y ^- , -Z-X ^- Y ⁺ , -X ^- Y ⁺ Or -Y,
X is a linker,
X ⁺ Is a positively charged linker,
X ^- Is a negatively charged linker,
Whether Y is a ribonucleoprotein or endonuclease containing a site-specific modified polypeptide, Y is a site-specific modified polypeptide, Y is a Cas9 ribonucleoprotein, or Y is a Cas9 protein , Y is a single guide RNA sequence (sgRNA) or Y is a dual guide RNA sequence comprising CRISPR RNA (crRNA) and transactivated crRNA (tracrRNA);
Y ⁺ Is a positively charged ribonucleoprotein or endonuclease containing a site-specific modified polypeptide, or Y ⁺ Is a positively charged site-specific modified polypeptide or Y ⁺ Is a positively charged Cas9 protein,
Y ^- Is a negatively charged ribonucleoprotein or endonuclease containing a site-specific modified polypeptide, or Y ^- Is a negatively charged site-specific modified polypeptide or Y ^- Is a negatively charged Cas9 ribonucleoprotein or Y ^- Is a negatively charged sgRNA or Y ^- Is a negatively charged dual guide RNA sequence comprising CRISPR RNA (crRNA) and transactivated crRNA (tracrRNA);
Z is absent or —C≡C—, —CH═CH—, —CH ₂ -, -CH ₂ -O-, -C (O) -N (R ^Four )-, -CH ₂ -S-, -CH ₂ -S (O)-, -CH ₂ -S (O) ₂ -, -CH ₂ -S (O) ₂ -N (R ^Four )-, -C (O) -O-, -CH ₂ -N (R ^Four )-, -CH ₂ -N (R ^Four ) -C (O)-, -CH ₂ -N (R ^Four ) -S (O) ₂ -, -CH ₂ -N (R ^Four ) -C (O) -O-, -CH ₂ -N (R ^Four ) -C (O) -N (R ^Four )-, -CH ₂ -O-C (O)-, -CH ₂ -O-C (O) -N (R ^Four )-, -CH ₂ -O-C (O) -O-, or aryl or heteroaryl, wherein the aryl or heteroaryl is optionally R ^Five Is replaced with
R ² Are —OH, —N _Three , -N (R ^Three ) ₂ , -N (R ^Three ) -C (O) -R ^Three , -N (R ^Three ) -C (O) -N (R ^Three ) ₂ , -N (R ^Three ) -C (O) -OR ^Three , -N (R ^Three ) -S (O) ₂ -R ^Three , Tetrazole, or triazole, wherein the tetrazole and triazole are optionally R ^Three Is replaced with
Each R ^Three Are independently -H,-(C ₁ -C _Five ) Alkyl, halo-substituted (C ₁ -C _Five ) Alkyl, halo-substituted (C _Three -C ₆ ) Cycloalkyl,-(C ₁ -C _Five ) Alkenyl,-(C ₁ -C _Five ) Alkynyl, halo-substituted- (C ₁ -C _Five ) Alkenyl, halo-substituted- (C ₁ -C _Five ) Alkynyl or (C _Three -C ₆ ) Cycloalkyl, the alkyl or cycloalkyl —CH ₂ -Groups are each independently -O-, -S-, and -N (R ^Four )-May be replaced with a heteroatom group selected from: -CH _Three Each independently represents —N (R ^Four ) ₂ , -OR ^Four , And -S (R ^Four ), Wherein the heteroatom group is separated by at least two carbon atoms;
Each R ^Four Are independently -H,-(C ₁ -C ₂₀ ) Alkyl,-(C ₁ -C ₂₀ ) Alkenyl,-(C ₁ -C ₂₀ ) Alkynyl or (C _Three -C ₆ ) 1-6 —CH of cycloalkyl, alkyl or cycloalkyl separated by said at least two carbon atoms ₂ -Groups are each independently -O-, -S-, or -N (R ^Four )-Optionally substituted with a heteroatom independently selected from -CH _Three Are each independently -N (R ^Four ) ₂ , -OR ^Four , And -S (R ^Four ), Wherein the heteroatom group is separated by at least two carbon atoms and the alkyl, alkenyl, alkynyl, and cycloalkyl may be substituted with a halo atom Often,
Each R ^Five Are independently -H, (C _Three -C ₂₀ ) Cycloalkyl,-(C ₁ -C ₆₀ ) Alkenyl,-(C ₁ -C ₆₀ ) Alkynyl or (C ₁ -C ₆₀ ) Alkyl, 1-6 —CH of said cycloalkyl ₂ A group, or 1-20 -CH of said alkyl ₂ -Groups are each independently -O-, -S-, and -N (R ^Four )-May be exchanged with a heteroatom independently selected from, wherein the heteroatom is separated by at least two carbon atoms and the alkyl —CH _Three Are each independently -N (R ^Four ) ₂ , -OR ^Four , And -S (R ^Four And the heteroatom group is separated by at least two carbon atoms; and the alkyl, alkenyl, alkynyl, and cycloalkyl may be substituted with a halo atom .
3. A compound according to embodiment 1 or 2, comprising R ² Is —NH—C (O) —CH _Three Said compound.
4). A compound according to any of embodiments 1-3, wherein R ¹ Wherein the compound is -Z-X + Y-.
5. A compound according to any of embodiments 1-3, wherein R ¹ Wherein the compound is -Z-X-Y +.
6). A compound according to any of embodiments 1-3, wherein R ¹ Wherein the compound is —Z—X—Y.
7). A compound according to embodiment 6, wherein R ¹ Wherein the compound is —Z—X—Y selected from the group consisting of L1 to L10:
Where
Each T independently does not exist or (C ₁ -C _Ten ) Alkylene, (C ₂ -C _Ten ) Alkenylene, or (C ₂ -C _Ten ) Alkynylene, wherein one or more carbon groups of T are each independently -O-, -S-, and -N (R ^Four )-May be replaced with a heteroatom group independently selected from, wherein the heteroatom group is separated by at least two carbon atoms, and the alkylene, alkenylene, and alkynylene are each independently 1 May be substituted with the above halo atoms,
Each Q is independently absent or C (O), C (O) -NR ^Four , NR ^Four -C (O), OC (O) -NR ^Four , NR ^Four -C (O) -O, -CH ₂ -, Heteroaryl, or O, S, S-S, S (O), S (O) ₂ And NR ^Four Wherein at least two carbon atoms are the heteroatom groups O, S, S—S, S (O), S (O) ₂ And NR ^Four From any other heteroatom group,
Each R ^Four Are independently -H,-(C ₁ -C ₂₀ ) Alkyl or (C _Three -C ₆ ) 1-6 —CH of said alkyl or cycloalkyl, which is cycloalkyl and separated by at least 2 carbon atoms ₂ The group is —O—, —S—, or —N (R ^Four )-And may be substituted with -CH _Three Are each independently -N (R ^Four ) ₂ , -OR ^Four , And -S (R ^Four A heteroatom group selected from: wherein the heteroatom group is separated by at least two carbon atoms, and the alkyl and cycloalkyl may be substituted with a halo atom;
Each m is independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40.
8). Embodiment 8. A compound according to embodiment 7, wherein each occurrence of Q is independently a heteroaryl selected from 1H-1,2,3-triazolyl, pyridinyl and 1,2,3,4-tetrazolyl. Said compound.
9. Embodiment 9. The compound according to embodiment 7 or 8, wherein X comprises a disulfide bond.
10. A compound according to embodiment 1, wherein the compound having the formula of formula (C-1), (C-2), (C-3) or (C4), or a pharmaceutically acceptable salt thereof:
Where
n is 1, 2 or 3,
W is absent or is a peptide,
L is-(TQ-TQ) _m −
Each T independently does not exist or (C ₁ -C _Ten ) Alkylene, (C ₂ -C _Ten ) Alkenylene or (C ₂ -C _Ten ) Alkynylene, wherein one or more carbon groups of T are each independently -O-, -S-, and -N (R ^Four )-May be replaced with a heteroatom group independently selected from, wherein the heteroatom group is separated by at least two carbon atoms, and each of alkylene, alkenylene, and alkynylene is independently one or more halo May be substituted with atoms,
Each Q is independently absent or C (O), C (O) -NR ^Four , NR ^Four -C (O), OC (O) -NR ^Four , NR ^Four -C (O) -O, -CH ₂ -, Heteroaryl, or O, S, S-S, S (O), S (O) ₂ And NR ^Four Wherein at least two carbon atoms are the heteroatom groups O, S, S—S, S (O), S (O) ₂ And NR ^Four From any other heteroatom group,
Each R ^Four Are independently -H,-(C ₁ -C ₂₀ ) Alkyl or (C _Three -C ₆ ) 1-6 —CH of said alkyl or cycloalkyl, which is cycloalkyl and separated by at least two carbon atoms ₂ The group is —O—, —S—, or —N (R ^Four )-, Which may be replaced with -CH of the alkyl _Three Are each independently -N (R ^Four ) ₂ , -OR ^Four , And -S (R ^Four ), Wherein the heteroatom group is separated by at least two carbon atoms, and the alkyl and cycloalkyl may be substituted with a halo atom,
Each m is independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40.
11. A compound according to embodiment 1, wherein the compound having the formula of formula (D-1) or (D-2), or a pharmaceutically acceptable salt thereof:
Where
n is 1, 2 or 3.
12 A compound according to embodiment 2, wherein the compound having formula (E), or a pharmaceutically acceptable salt thereof:
Where
n is 1, 2 or 3,
W is absent or is a peptide,
L is-(TQ-TQ) _m −
Where
Each T independently does not exist or (C ₁ -C _Ten ) Alkylene, (C ₂ -C _Ten ) Alkenylene or (C ₂ -C _Ten ) Alkynylene, wherein one or more carbon groups of T are each independently -O-, -S-, and -N (R ^Four )-May be substituted with a heteroatom group independently selected from-, said heteroatom group being separated by at least two carbon atoms;
Each Q is independently absent or C (O), C (O) -NR ^Four , NR ^Four -C (O), OC (O) -NR ^Four , NR ^Four -C (O) -O, -CH ₂ -, Heteroaryl, or O, S, S-S, S (O), S (O) ₂ And NR ^Four Wherein at least two carbon atoms are the heteroatom groups O, S, S—S, S (O), S (O) ₂ And NR ^Four From any other heteroatom group,
Each R ^Four Are independently -H,-(C ₁ -C ₂₀ ) Alkyl or (C _Three -C ₆ ) 1-6 —CH of said alkyl or cycloalkyl, which is cycloalkyl and separated by at least two carbon atoms ₂ -Groups are each independently -O-, -S-, or -N (R ^Four )-And may be replaced with -CH of the alkyl _Three Is -N (R ^Four ) ₂ , -OR ^Four , And -S (R ^Four ), Wherein the heteroatom group is separated by at least two carbon atoms, and the alkyl and cycloalkyl may be substituted with a halo atom, and
Each m is independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40.
13. A compound according to embodiment 2, wherein the compound having the formula of formula (F-1) or (F-2), or a pharmaceutically acceptable salt thereof:
Where
n is selected from 1, 2 and 3.
14 Embodiment 14. The compound according to any one of Embodiments 10 to 13, wherein n is 3.
15. Embodiment 12. A compound according to embodiment 12 having the formula
Or a pharmaceutically acceptable salt thereof.
16. Embodiment 13. The compound according to embodiment 10 or 12, wherein W is a peptide that is an endosomal lytic peptide or a nuclear localization peptide.
17. A compound according to any one of the preceding embodiments, wherein Y is
(1) a first element comprising a recognition element comprising either a dual guide RNA sequence comprising CRISPR RNA (crRNA) and transactivated crRNA (tracrRNA) or a single guide RNA sequence (sgRNA), The sequence, when expressed, directs sequence specific binding to the target sequence of the Cas9 ribonucleoprotein, wherein the first element optionally comprises one or more endosomal escape agents;
(2) a Cas9 ribonucleoprotein comprising a Cas9 protein and optionally a second element comprising one or more nuclear localization sequences (NLS) and optionally one or more fluorescent proteins and one or more endosomal escape agents. ,
The compound wherein the first element is associated with the second element.
18. Embodiment 18. The compound according to embodiment 17, wherein the target sequence is a eukaryotic target sequence.
19. Embodiment 18. The compound of embodiment 17, wherein the first element comprises a dual guide RNA sequence comprising CRISPR RNA (crRNA) and transactivated crRNA (tracrRNA), each of the rest of the compounds independently Said compound linked to said Cas9 ribonucleoprotein via one or more interactions with a tracrRNA sequence or crRNA.
20. Embodiment 20. The compound according to embodiment 19, wherein the tracrRNA is optionally chemically modified.
21. Embodiment 19. The compound of embodiment 19 or 20, wherein the tracrRNA is at least about 75%, at least about 80%, at least about 80%, Comprising sequences having nucleic acid sequence identity of 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100%, said sequences optionally comprising the first three Said compound comprising a 2'-O-methyl or 2'-F modification in the base.
22. Embodiment 22. The compound according to any one of Embodiments 19 to 21, wherein the crRNA is optionally chemically modified.
23. The compound of any one of embodiments 19-21, wherein the crRNA is at least about 75%, at least about 80%, at least about 85%, at least about 90% relative to a sequence selected from: Wherein said compound comprises a sequence having a nucleotide sequence identity of at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100%:
PCSK9 crRNA sequence 1:
GGUGCUAGCCUUGCGUUCCGGUUUUAAGCUAUGCUG (SEQ ID NO: 885), optionally including 2'-O-methyl or 2'-F modifications in the first three of the last four bases (GCU),
PCSK9 crRNA sequence 2:
CUGUCUCGGGUGCUUCGGCCGUUUUAAGAGCUAUUGCUG (SEQ ID NO: 886), optionally including 2'-O-methyl or 2'-F modifications in the first three of the last four bases (GCU),
PCSK9 crRNA sequence 3:
GCCGUCCUCCUCGGAACCGCAGUUUUAAGAGCUAUUGCUG (SEQ ID NO: 887), optionally including 2'-O-methyl or 2'-F modifications in the first 3 of the last 4 bases (GCU),
PCSK9 crRNA sequence 4:
GGACGAGGACGGCGACUACGGGUUUUAAGAGCUAUUGCUG (SEQ ID NO: 888), optionally including 2'-O-methyl or 2'-F modifications in the first 3 of the last 4 bases (GCU),
PCSK9 crRNA sequence 5:
ACACCCGGGGAAAUCGAGGGCGUUUUAAGAGCUAUUGCUG (SEQ ID NO: 889), optionally including 2′-O-methyl or 2′-F modifications in the first three of the last four bases (GCU),
PCSK9 crRNA sequence 6:
CGACUUCGAGAAUGUGCCCCGUUUUAAGAGCUAUUGCUG (SEQ ID NO: 890), optionally including 2'-O-methyl or 2'-F modifications in the first 3 of the last 4 bases (GCU),
PCSK9 crRNA sequence 7:
GAGUGACCACCGGGAAAUCGGUUUUAAGAGCUAUUGCUG (SEQ ID NO: 891), optionally including 2′-O-methyl or 2′-F modifications in the first 3 of the last 4 bases (GCU),
PCSK9 crRNA sequence 8:
CUCGGGCACAUCUCUCGAAGUGUUUAAGAGCUAUUGCUG (SEQ ID NO: 892), optionally including 2′-O-methyl or 2′-F modifications in the first three of the last four bases (GCU),
PCSK9 crRNA sequence 9:
GGAAGCCAGGAAGAAGGCCCAGUUUAGAGCUAUUGCUG (SEQ ID NO: 893), optionally including 2′-O-methyl or 2′-F modifications in the first three of the last four bases (GCU),
PCSK9 crRNA sequence 10:
UCUUUGCCCCAGAGCAUCCCGGUUUUAAGAGCUAUUGCUG (SEQ ID NO: 894), optionally including 2'-O-methyl or 2'-F modifications in the first 3 of the last 4 bases (GCU),
PCSK9 crRNA sequence 11:
CUAGGAGAUAACACCUCCCACCGUUUUAGGAGCUAUUGCUG (SEQ ID NO: 895), optionally including 2'-O-methyl or 2'-F modifications in the first 3 of the last 4 bases (GCU).
24. Embodiment 18. The compound of embodiment 17, wherein the first element comprises a single guide RNA sequence (sgRNA) and the remainder of the compound is linked to the Cas9 ribonucleoprotein via one or more interactions with sgRNA. Said compound.
25. Embodiment 25. A compound according to embodiment 24, wherein the sgRNA comprises at least 20 nucleotides.
26. Embodiment 25. A compound according to embodiment 24, wherein the sgRNA comprises at least 8 nucleotides.
27. The compound of embodiment 24, wherein the degree of complementarity between the sgRNA and its corresponding target sequence is at least 50%, 60 when optimally aligned using an appropriate alignment algorithm %, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, Said compound which is 98%, 99% or 100%.
28. 25. The compound of any one of embodiments 24, wherein the sgRNA is at least about 75%, at least about 80%, at least about 85%, at least about a sequence selected from the group consisting of: 90%, at least about 95%, at least about 96% said compound having at least about 97 percent, at least about 98 percent, at least about 99 percent or 100 percent nucleotide sequence identity:
PCSK9 single guide RNA sequence 1
GGUGCUAGCCUUGCGUUCCGGUUUAAGAGCUAUUGCUGGAAACAGCAAUAGCAAGUUUAAAAUAGAGCUAGUCCCGUUACACACUUGAAAAAAGUGGCACCGAGUCGGUUGCUUUUUUUUUU
PCSK9 single guide RNA sequence 2
CUGUGUCGGGGUGCUUCGGCCGUUUAAGAGCUAUUGUGGAAACAGCAUGAGCAAGUUUAAAAUAGGCUAUGCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUUGCUUUUUUUUUUU
PCSK9 single guide RNA sequence 3
GCCGUCCUCCUCGGAACCGCAGUUUAAGAGCUAUUGCUGGAAACAGCAAUAGCAAGUUUAAAAUAGGGCUAGUCCCGUUAUCAACUUGAAAAAGUGGCACCACCAGUCGGUCUUUUUU (sequence number 898)
PCSK9 single guide RNA sequence 4
GGACGAGGACGGCGACUACGGGUAUUAAGAGCUAUUGCUGGAAACAGCAAUAGCAAGUUUAAAUAAGGGCUAGUCCCGUUAUCAACUUGAAAAAGUGGCACCGAGGUCGUGUCUUUUUU (sequence number 899)
PCSK9 single guide RNA sequence 5
ACACCCGGGGAAAUCGAGGGCGUAUUAAGAGCUAUUGCUGGAAACAGCAAUAGCAAGUUUAAAUAAGGGCUAGUCCCGUUAUCAACUUGAAAAAGUGGCACCAGAGUCGGGUCUUUUUU (sequence number 900)
PCSK9 single guide RNA sequence 6
CGACUUCGAGAAUGUGCCCCGGUUUAAGAGCUAUUGCUGGAAACAGCAAUAGCAAGUUUAAAUAAGGGCUAGUCCCGUUAUCAACUUGAAAAAGUGGCACCGAGGUCGUGUCUUUUUU (sequence number 901)
PCSK9 single guide RNA sequence 7
GAUGACACCACCGGGAAAUCGGGUUUAAGAGCUAUUGCUGGAAACAGCAAUAGCAAGUUUAAAUAAGGGCUAGUCCCGUUAUCAACUUGAAAAAGUGGCACCGAGGUCGUGUCUUUUUU (sequence number 902)
PCSK9 single guide RNA sequence 8
CUCGGGCACAUUCUCGAAGUGUUUAAGAGCUAUUGCUGGAAACAGCAAUAGCAAGUUUAAAUAAGGGCUAGUCCCGUUAUCAACUUGAAAAAGUGGCACCAGAGUCGUGUCUUUUUU (sequence number 903)
PCSK9 single guide RNA sequence 9
GGAAGCCAGGAAGAAAGGCCAGUUUAAGAGCUAUUGCUGGAAACAGCAUGAGCAAGUUUAAAAUAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGGUCGUGUUUUUUU (sequence number 904),
PCSK9 single guide RNA sequence 10
UCUUUGCCCCAGAGCAUCCCGGGUUUAAGAGCUAUUGCUGGAAACAGCAUAGCAAGUUUAAAUAAGGGCUAGUCCCGUUAUCAACUUGAAAAAGUGGCACCAGAGUCGUGUCUUUUUU (sequence number 905)
PCSK9 single guide RNA sequence 11
CUAGGAGAUAACACCUCCCACCGUUUAAGAGCUAUUGCUGGAAACAGCAAUAGCAAGUUUAAAAUAGGCUAGUCCGUUAUCAACUGUAAAAAUGGCACCAGGAUCGGUGCUUUUUU (sequence number 906)
EMX1 single guide RNA sequence
GUCACCUCCAAUGACAGUAGGGGUAUUAAGAGCUAUUGCUGGAAACAGCAAUAGCAAGUUUAAAUAAGGGCUAGUCCCGUUAUCAACUUGAAAAAGUGGCACCAGAGUCGUGUCUUUUUU (sequence number 907)
ROSA26 single guide RNA sequence
CGAACCCUACACAUUCAACGGUUUAAGAGCUAUUGCUGGAAACAGCAUAGGCAAGUUUAAAAUAGGCUAGUCCGUUAUCAACUGUAAAAAUGGCACCCGAGUCGGUUGCUUUUUU (sequence number 908)
Here, the sequence is optionally chemically modified.
29. Embodiment 29. The compound according to any one of embodiments 24 to 28, wherein the Y comprises a sgRNA optionally chemically modified.
30. Embodiment 30. The compound according to any one of Embodiments 1 to 29, wherein the Y further comprises a fluorescent probe.
31. Embodiment 30. The compound according to embodiment 30, wherein the fluorescent probe is an mCherry sequence (SEQ ID NO: 915).
32. The compound of any one of the preceding embodiments, wherein Y comprises one or more NLS.
33. Embodiment 33. The compound according to embodiment 32, wherein each of the NLS comprises an amino acid sequence selected from the group consisting of:
PKKKRKV (SEQ ID NO: 830),
KRPAATKKAGQAKKKK (SEQ ID NO: 831),
PAAKRVKLD (SEQ ID NO: 832),
RQRRNELKRSP (SEQ ID NO: 833),
NQSSNFGPMKGNFNFGSSGYPYGGGGQYFAKPRNQGGY (SEQ ID NO: 834),
RMRIXFKNKKGKDTAELRRRRVEVSVVERLKAKKDEQILKRRNV (SEQ ID NO: 835),
VSRKRPRP (SEQ ID NO: 836),
PPKKARED (SEQ ID NO: 837),
PQPKKKKPL (SEQ ID NO: 838),
SALIKKKKMAP (SEQ ID NO: 839),
DRLRR (SEQ ID NO: 840),
PKQKKRK (SEQ ID NO: 841),
RKLKKKIKKL (SEQ ID NO: 842),
REKKKFLKRR (SEQ ID NO: 843),
KRKGDEVDGVDEVAKKKSKK (SEQ ID NO: 844),
RKCLQAGMNLEARKTKK (SEQ ID NO: 845),
MAPKKKRKVGIHRGVP (SEQ ID NO: 1035), and
PKKRKVEDPKKKRKVD (SEQ ID NO: 1036).
34. Embodiment 34. The compound according to embodiment 33, wherein each of the NLSs comprises the amino acid sequence PKKRKV (SEQ ID NO: 830).
35. Embodiment 35. A compound according to embodiment 34, comprising two NLS, each comprising the amino acid sequence PKKKRRKV (SEQ ID NO: 830).
36. The compound of any one of the preceding embodiments, wherein Y is S.I. aureus, S .; pneumoniae, S .; pyogenes, S .; thermophilus, N.M. meningitidis or A.M. At least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98 percent, at least about at least about Cas9 protein from ebreus Said compound comprising a Cas9 protein that is 99 percent, or 100 percent amino acid sequence identity.
37. The compound of any one of the preceding embodiments, wherein Y is at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about Type II Cas9 protein The compound comprising a Cas9 protein that is about 95%, at least about 90%, at least about 95%, at least about 96 percent, at least about 97 percent, at least about 98 percent, at least about 99 percent, or 100 percent amino acid sequence identity .
38. The compound according to any one of the preceding embodiments, wherein Y is an amino acid at positions 7 to 166 or 731 to 1003 of SEQ ID NO: 8, or a sequence according to SEQ ID NOs: 1 to 7, 9 to 829 A Cas9 protein that is at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99 percent, or 100 percent amino acid sequence identity to the corresponding amino acid. Said compound comprising.
39. The compound of any one of the preceding embodiments, wherein Y is at least about 75%, at least about 80%, relative to the amino acids at positions 7-166, or 731-1003 of SEQ ID NO: 8. A Cas9 protein that is at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99 percent, or 100 percent amino acid sequence identity Said compound.
40. 264. The compound of any one of the previous embodiments, wherein Y is at least about 75% relative to motif 9, 2, 3, and 4 of the Cas9 amino acid sequence of any of SEQ ID NOs: 260-263. At least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99 percent, or 100 percent amino acid sequence identity. Said compound comprising a Cas9 protein having at least 4 motifs in the sequence having it.
41. The compound of any one of the preceding embodiments, wherein Y is S.I. pyogenesCas9 (wild type) (SEQ ID NO: 848), S. pyogenesCas9-mutant M1C (SEQ ID NO: 849), S. cerevisiae. pyogenesCas9-mutant M1C & C80S (SEQ ID NO: 850), S. pyogenesCas9 nickase mutant D10A (SEQ ID NO: 851), S. pylori. pyogenesCas9 nickase mutant H840A (SEQ ID NO: 852), S. pyogenesCas9 nickase mutants E923P & T924P (SEQ ID NO: 853), Acidovorax ebreus (Acidovorax ebreus) Cas9 (SEQ ID NO: 854), acid mine drainage fungus Ga0052161_JGI · Cas9 (SEQ ID NO: 855), at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about a sequence selected from pyogenesCas9 null mutant D10A & H840A (SEQ ID NO: 1027) and uranium mining fungus FW106_JGICas9 (sequence 856) The compound comprising a Cas9 protein having about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99 percent, or 100 percent amino acid sequence identity.
42. The compound according to any one of the previous embodiments, which is capable of binding to a receptor present on hepatocytes.
43. 43. The compound according to embodiment 42, wherein the receptor present in the hepatocytes is an asialoglycoprotein receptor.
44. The compound of any one of the preceding embodiments, further comprising an endosome escape agent.
45. The compound of any one of the previous embodiments, wherein said Y further comprises an endosome escape agent.
46. 46. The compound according to embodiment 44 or 45, wherein the endosome escape agent is selected from the group consisting of a lysosome agonist, a cell penetrating peptide, a fusogenic peptide, a pore-forming agent, and a proton sponge agent.
47. 47. The compound according to embodiment 46, wherein the endosomal escape agent is peptide ppTG21: GLFHALLHLLHSLWHLLLLHA (SEQ ID NO: 1012).
48. 48. A composition comprising the compound of any one of embodiments 1-47 and an endosome escape agent, wherein the compound and the endosome escape agent are co-incubated to form the composition. .
49. 49. The composition of embodiment 48, wherein the endosome escape agent is selected from the group consisting of lysosome agonists, cell penetrating peptides, fusogenic peptides, pore formers, and proton sponge agents. .
50. 50. The composition of embodiment 49, wherein the endosomal escape agent is peptide ppTG21: GLFHALLHLLHSLWHLLHA (SEQ ID NO: 1012).
51. A pharmaceutical composition comprising a compound according to any one of the preceding embodiments and a pharmaceutically acceptable carrier, excipient or diluent.
52. 52. A method of treating a liver disease or condition or a liver regulatory disease or condition in a subject, comprising administering to the subject a therapeutically effective amount of the pharmaceutical composition of embodiment 51.
53. 53. The method of embodiment 52, wherein the disease or condition is inherited angioedema, familial tyrosinemia type I, Alagille syndrome, alpha-1-antitrypsin deficiency, bile acid synthesis and metabolic disorders, biliary tract Atresia, cystic fibrosis liver disease, idiopathic neonatal hepatitis, mitochondrial hepatopathy, progressive familial intrahepatic cholestasis, primary sclerosing cholangitis, transthyretin amyloidosis, hemophilia, homozygous familial With hypercholesterolemia, familial chylomicronemia, hyperlipidemia, steatohepatitis, nonalcoholic steatohepatitis (NASH), nonalcoholic fatty liver disease (NAFLD), hyperglycemia, and type II diabetes The method as described above, also selected from the group consisting of diseases with abnormally high liver glucose production.
54. 54. The method of embodiment 53, wherein the disease or condition is hyperlipidemia, nonalcoholic steatohepatitis (NASH), or nonalcoholic steatohepatitis (NAFLD).
55. 52. A method of selectively modulating transcription of target DNA in a subject's liver cells, comprising administering to said subject the pharmaceutical composition according to embodiment 51.
56. 56. The method of embodiment 55, wherein the target DNA is hereditary angioedema, familial tyrosinemia type I, Alagille syndrome, alpha-1-antitrypsin deficiency, bile acid synthesis and metabolic disorders, biliary atresia Disease, cystic fibrosis liver disease, idiopathic neonatal hepatitis, mitochondrial hepatopathy, progressive familial intrahepatic cholestasis, primary sclerosing cholangitis, transthyretin amyloidosis, hemophilia, homozygous familial high Similar to cholesterolemia, familial chylomicronemia, hyperlipidemia, steatohepatitis, nonalcoholic steatohepatitis (NASH), nonalcoholic fatty liver disease (NAFLD), hyperglycemia, and type II diabetes Said method relating to a disease or condition selected from the group consisting of diseases with abnormally high liver glucose production.
57. 56. The method of embodiment 55, wherein the target DNA is a PCSK9 gene.
58. 49. A method of editing a nucleic acid molecule encoding a protein associated with a liver disease or condition in a subject, the method comprising administering to the subject a pharmaceutical composition according to embodiment 48.
59. 58. The method of embodiment 57, wherein said protein is hereditary angioedema, familial tyrosinemia type I, Alagille syndrome, alpha-1-antitrypsin deficiency, bile acid synthesis and metabolic disorders, biliary atresia, cyst Fibrosis liver disease, idiopathic neonatal hepatitis, mitochondrial hepatopathy, progressive familial intrahepatic cholestasis, primary sclerosing cholangitis, transthyretin amyloidosis, hemophilia, homozygous familial hypercholesterolemia , Familial chylomicronemia, hyperlipidemia, steatohepatitis, nonalcoholic steatohepatitis (NASH), nonalcoholic fatty liver disease (NAFLD), hyperglycemia, and abnormalities similar to type II diabetes Said method relating to a liver disease or condition selected from the group consisting of diseases with high liver glucose production.
60. 52. A method of modulating the expression level of at least one gene product associated with a liver disease or condition in a subject, comprising administering to the subject the pharmaceutical composition of embodiment 51.
61. 61. The method of embodiment 60, wherein the gene product is hereditary angioedema, familial tyrosinemia type I, Alagille syndrome, alpha-1-antitrypsin deficiency, bile acid synthesis and metabolic abnormalities, biliary atresia Disease, cystic fibrosis liver disease, idiopathic neonatal hepatitis, mitochondrial hepatopathy, progressive familial intrahepatic cholestasis, primary sclerosing cholangitis, transthyretin amyloidosis, hemophilia, homozygous familial high Similar to cholesterolemia, familial chylomicronemia, hyperlipidemia, steatohepatitis, nonalcoholic steatohepatitis (NASH), nonalcoholic fatty liver disease (NAFLD), hyperglycemia, and type II diabetes Said method relating to a liver disease or condition selected from the group consisting of diseases with abnormally high liver glucose production.
62. 62. The method of embodiment 61, wherein the method modulates the level of low density lipoprotein (LDL).
63. 62. The method of embodiment 61, wherein the method modulates blood cholesterol levels in the subject.
64. 64. The method of embodiment 63, wherein the method reduces blood cholesterol levels in the subject.
65. A composition comprising a ribonucleoprotein and an endosome escape agent.
66. 66. The composition of embodiment 65, wherein the ribonucleoprotein is a Cas9 ribonucleoprotein or a Cpfl ribonucleoprotein.
67. Embodiment 67. The composition according to embodiment 66, wherein the ribonucleoprotein is a Cas9 ribonucleoprotein.
68. 68. The composition of embodiment 67, wherein the Cas9 ribonucleoprotein and the endosomal escape agent are co-incubated.
69. Embodiment 69. The composition of embodiment 67 or 68, wherein the Cas9 ribonucleoprotein is
(1) a first element comprising a recognition element comprising either a dual guide RNA sequence comprising CRISPR RNA (crRNA) and transactivated crRNA (tracrRNA) or a single guide RNA sequence (sgRNA), The guide sequence, when expressed, directs sequence specific binding to the target sequence of the Cas9 ribonucleoprotein, said first element optionally comprising a first element comprising one or more endosomal escape agents;
(2) comprising a second element comprising a Cas9 protein and optionally one or more nuclear localization sequences (NLS) and optionally one or more fluorescent proteins, and one or more endosomal escape agents;
The composition wherein the first element is associated with the second element.
70. 70. The composition of claim 69, wherein the target sequence is a eukaryotic target sequence.
71. 70. The composition of embodiment 69, wherein the first element comprises a dual guide RNA sequence comprising CRISPR RNA (crRNA) and transactivated crRNA (tracrRNA).
72. 72. The composition of embodiment 71, wherein the tracrRNA is optionally chemically modified.
73. The composition according to embodiment 71 or 72, wherein the tracrRNA is at least about 75%, at least about 80%, at least about 85%, at least about 85%, at least about 85%, Comprising about 95%, or at least about 95%, at least about 96 percent, at least about 97 percent, at least about 98 percent, at least about 99 percent, or 100 percent nucleotide sequence identity, said sequence optionally comprising: Said composition comprising a 2'-O-methyl or 2'-F modification in the first three bases.
74. 74. The composition according to any one of embodiments 71 to 73, wherein the crRNA is optionally chemically modified.
75. The composition according to any one of embodiments 71-73, wherein said crRNA is at least about 75%, at least about 80%, at least about 85%, at least about 90 relative to a sequence selected from: The composition comprising a sequence having a nucleotide sequence identity of%, at least about 95%, about 96 percent, at least about 97 percent, at least about 98 percent, at least about 99 percent, or 100 percent:
PCSK9 crRNA sequence 1:
GGUGCUAGCCUUGCGUUCGGUUUUAAGAGCUAUUGCUG (SEQ ID NO: 885), optionally including 2'-O-methyl or 2'-F modifications in the first 3 bases (GCU) of the last 4 bases
PCSK9 crRNA sequence 2:
CGUGCUCGGGUGCUUCGGCCGUUUUAGAGCUAUUGCUG (SEQ ID NO: 886), optionally including 2'-O-methyl or 2'-F modifications in the first 3 bases (GCU) of the last 4 bases
PCSK9 crRNA sequence 3:
GCCGUCCUCCCUGGAACCGCAGUUUUAAGAGCUAUUGCUG (SEQ ID NO: 887), optionally including 2'-O-methyl or 2'-F modifications in the first 3 bases (GCU) of the last 4 bases
PCSK9 crRNA sequence 4:
GGACGAGGACGGCGACUACGGGUUUUAAGAGCUAUUGCUG (SEQ ID NO: 888), optionally including 2'-O-methyl or 2'-F modifications in the first 3 bases (GCU) of the last 4 bases
PCSK9 crRNA sequence 5:
ACCCACCGGGAAAUCGAGGGGGUUUUAAGAGCUAUUGCUG (SEQ ID NO: 889), optionally including 2'-O-methyl or 2'-F modifications in the first 3 bases (GCU) of the last 4 bases
PCSK9 crRNA sequence 6:
CGACUUCGAGAAUGUGCCCCGUUUUAAGAGCUAUUGCUG (SEQ ID NO: 890), optionally including 2'-O-methyl or 2'-F modifications in the first 3 bases (GCU) of the last 4 bases
PCSK9 crRNA sequence 7:
GAGUGACCACCGGGAAAUCGGUUUUAAGAGCUAUUGCUG (SEQ ID NO: 891), optionally including 2'-O-methyl or 2'-F modifications in the first 3 bases (GCU) of the last 4 bases
PCSK9 crRNA sequence 8:
CUCGGGCACAUUCUCGAAGUGUUUAAGAGCUAUUGCUG (SEQ ID NO: 892), optionally including 2'-O-methyl or 2'-F modifications in the first 3 bases (GCU) of the last 4 bases
PCSK9 crRNA sequence 9:
GGAAGCCAGGAAGAAGGCCAGUUUAGAGCUAUUGCUG (SEQ ID NO: 893), optionally including 2'-O-methyl or 2'-F modifications in the first 3 bases (GCU) of the last 4 bases
PCSK9 crRNA sequence 10:
UCUUUGCCCCAGAGUCUCCCGGGUUUAGAGCUAUUGCUG (SEQ ID NO: 894), optionally including 2'-O-methyl or 2'-F modifications in the first 3 bases (GCU) of the last 4 bases
PCSK9 crRNA sequence 11:
CUAGGAGAUAACACCUCCCACCGUUUUAGGAGCUAUUGCUG (SEQ ID NO: 895), optionally including 2'-O-methyl or 2'-F modifications in the first 3 bases (GCU) of the last 4 bases
76. 70. The composition of embodiment 69, wherein the first element comprises a single guide RNA sequence (sgRNA).
77. Embodiment 76. The composition of embodiment 76, wherein the sgRNA comprises at least 20 nucleotides.
78. Embodiment 76. The composition of embodiment 76, wherein the sgRNA comprises at least 8 nucleotides.
79. 77. The composition of embodiment 76, wherein the degree of complementarity between the sgRNA and its corresponding target sequence is at least 50%, 60 when optimally aligned using an appropriate alignment algorithm. %, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, The composition is 98%, 99%, or 100%.
80. The composition of embodiment 76, wherein the sgRNA is at least about 75 percent, at least about 80 percent, at least about 85 percent, at least about 90 percent relative to a sequence selected from the group consisting of: The composition having a nucleotide sequence identity of at least about 95 percent, at least about 96 percent, at least about 97 percent, at least about 98 percent, at least about 99 percent, or 100 percent:
PCSK9 guide RNA sequence 1
GGUGCUAGCCUUGCGUUCCGGUUUAAGAGCUAUUGCUGGAAACAGCAAUAGCAAGUUUAAAAUAGAGCUAGUCCCGUUACACACUUGAAAAAAGUGGCACCGAGUCGGUUGCUUUUUUUUUU
PCSK9 guide RNA sequence 2
CUGUGUCGGGGUGCUUCGGCCGUUUAAGAGCUAUUGUGGAAACAGCAUGAGCAAGUUUAAAAUAGGCUAUGCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUUGCUUUUUUUUUUU
PCSK9 guide RNA sequence 3
GCCGUCCUCCUCGGAACCGCAGUUUAAGAGCUAUUGCUGGAAACAGCAAUAGCAAGUUUAAAAUAGGGCUAGUCCCGUUAUCAACUUGAAAAAGUGGCACCACCAGUCGGUCUUUUUU (sequence number 898)
PCSK9 guide RNA sequence 4
GGACGAGGACGGCGACUACGGGUAUUAAGAGCUAUUGCUGGAAACAGCAAUAGCAAGUUUAAAUAAGGGCUAGUCCCGUUAUCAACUUGAAAAAGUGGCACCGAGGUCGUGUCUUUUUU (sequence number 899)
PCSK9 guide RNA sequence 5
ACACCCGGGGAAAUCGAGGGCGUAUUAAGAGCUAUUGCUGGAAACAGCAAUAGCAAGUUUAAAUAAGGGCUAGUCCCGUUAUCAACUUGAAAAAGUGGCACCAGAGUCGGGUCUUUUUU (sequence number 900)
PCSK9 guide RNA sequence 6
CGACUUCGAGAAUGUGCCCCGGUUUAAGAGCUAUUGCUGGAAACAGCAAUAGCAAGUUUAAAUAAGGGCUAGUCCCGUUAUCAACUUGAAAAAGUGGCACCGAGGUCGUGUCUUUUUU (sequence number 901)
PCSK9 guide RNA sequence 7
GAUGACACCACCGGGAAAUCGGGUUUAAGAGCUAUUGCUGGAAACAGCAAUAGCAAGUUUAAAUAAGGGCUAGUCCCGUUAUCAACUUGAAAAAGUGGCACCGAGGUCGUGUCUUUUUU (sequence number 902)
PCSK9 guide RNA sequence 8
CUCGGGCACAUUCUCGAAGUGUUUAAGAGCUAUUGCUGGAAACAGCAAUAGCAAGUUUAAAUAAGGGCUAGUCCCGUUAUCAACUUGAAAAAGUGGCACCAGAGUCGUGUCUUUUUU (sequence number 903)
PCSK9 guide RNA sequence 9
GGAAGCCAGGAAGAAAGGCCAGUUUAAGAGCUAUUGCUGGAAACAGCAUGAGCAAGUUUAAAAUAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGGUCGUGUUUUUUU (sequence number 904),
PCSK9 guide RNA sequence 10
UCUUUGCCCCAGAGCAUCCCGGGUUUAAGAGCUAUUGCUGGAAACAGCAUAGCAAGUUUAAAUAAGGGCUAGUCCCGUUAUCAACUUGAAAAAGUGGCACCAGAGUCGUGUCUUUUUU (sequence number 905)
PCSK9 guide RNA sequence 11
CUAGGAGAUAACACCUCCCACCGUUUAAGAGCUAUUGCUGGAAACAGCAAUAGCAAGUUUAAAAUAGGCUAGUCCGUUAUCAACUGUAAAAAUGGCACCAGGAUCGGUGCUUUUUU (sequence number 906)
EMX1 guide RNA sequence
GUCACCUCCAAUGACAGUAGGGGUAUUAAGAGCUAUUGCUGGAAACAGCAAUAGCAAGUUUAAAUAAGGGCUAGUCCCGUUAUCAACUUGAAAAAGUGGCACCAGAGUCGUGUCUUUUUU (sequence number 907)
ROSA26 guide RNA sequence
CGAACCCUACACAUUCAACGGUUUAAGAGCUAUUGCUGGAAACAGCAUAGGCAAGUUUAAAAUAGGCUAGUCCGUUAUCAACUGUAAAAAUGGCACCCGAGUCGGUUGCUUUUUU (sequence number 908)
Here, the sequence is optionally chemically modified.
81. 81. The composition of any one of embodiments 76-80, wherein the sgRNA is optionally chemically modified.
82. 82. The composition according to any one of embodiments 67 to 81, wherein the Cas9 ribonucleoprotein further comprises a fluorescent probe.
83. 83. The composition of embodiment 82, wherein the fluorescent probe is an mCherry sequence (SEQ ID NO: 915).
84. 84. The composition according to any one of embodiments 67 to 83, wherein the composition comprises one or more NLS.
85. 84. The composition according to any one of embodiments 67 to 83, wherein the Cas9 ribonucleoprotein comprises one or more NLS.
86. Embodiment 88. The composition of embodiment 84 or 85, wherein each of the NLSs comprises an amino acid sequence selected from the group consisting of:
PKKKRKV (SEQ ID NO: 830),
KRPAATKKAGQAKKKK (SEQ ID NO: 831),
PAAKRVKLD (SEQ ID NO: 832),
RQRRNELKRSP (SEQ ID NO: 833),
NQSSNFGPMKGNFNFGSSGYPYGGGGQYFAKPRNQGGY (SEQ ID NO: 834),
RMRIXFKNKKGKDTAELRRRRVEVSVVERLKAKKDEQILKRRNV (SEQ ID NO: 835),
VSRKRPRP (SEQ ID NO: 836),
PPKKARED (SEQ ID NO: 837),
PQPKKKKPL (SEQ ID NO: 838),
SALIKKKKMAP (SEQ ID NO: 839),
DRLRR (SEQ ID NO: 840),
PKQKKRK (SEQ ID NO: 841),
RKLKKKIKKL (SEQ ID NO: 842),
REKKKFLKRR (SEQ ID NO: 843),
KRKGDEVDGVDEVAKKKSKK (SEQ ID NO: 844),
RKCLQAGMNLEARKTKK (SEQ ID NO: 845),
MAPKKKRKVGIHRGVP (SEQ ID NO: 1035), and
PKKRKVEDPKKKRKVD (SEQ ID NO: 1036).
87. 90. The composition of embodiment 86, wherein each of the NLSs comprises the amino acid sequence PKKKRKV (SEQ ID NO: 830).
88. 90. The composition of embodiment 87, comprising two NLS, each of which comprises the amino acid sequence PKKKRRKV (SEQ ID NO: 830).
89. 89. The composition according to any one of embodiments 67 to 88, wherein the Cas9 ribonucleoprotein is an S. cerevisiae protein. aureus, S .; pneumoniae, S .; pyogenes, S .; thermophilus, or N.P. at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98 percent, at least about at least about Cas9 protein from meningitidis The composition comprising Cas9 protein that is 99 percent, or 100 percent amino acid sequence identity.
90. 90. The composition of any one of embodiments 67-89, wherein the Cas9 ribonucleoprotein is at least about 75%, at least about 80%, at least about 85%, at least about Type II Cas9 protein The composition comprising Cas9 protein that is about 90%, at least about 95%, at least about 96 percent, at least about 97 percent, at least about 98 percent, at least about 99 percent, or 100 percent amino acid sequence identity.
91. Embodiment 91. The composition according to any one of embodiments 67 to 90, wherein the Cas9 ribonucleoprotein is an amino acid at positions 7 to 166 or 731 to 1003 of SEQ ID NO: 8, or SEQ ID NOs: 1 to 7, 9 to 829 relative to the corresponding amino acid of at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96 percent, at least about 97 percent, at least about 98 The composition comprising Cas9 ribonucleoprotein that is percent, at least about 99 percent, or 100 percent amino acid sequence identity.
92. 92. The composition of embodiment 91, wherein the Cas9 ribonucleoprotein is at least about 75%, at least about 80%, at least relative to amino acids at positions 7-166, or 731-1003 of SEQ ID NO: 8 The composition comprising a Cas9 protein that is about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99 percent, or 100 percent amino acid sequence identity object.
93. 93. The composition of embodiment 92, wherein the Cas9 ribonucleoprotein is at least about 75%, at least about about 75% of motif 9, 2, 3, and 4 of the Cas9 amino acid sequence of any of SEQ ID NOs: 260-263. Within a sequence having 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99 percent, or 100 percent amino acid sequence identity The composition comprising a Cas9 protein having at least 4 motifs.
94. 94. The composition of any one of embodiments 67 to 93, wherein the Cas9 ribonucleoprotein is S. cerevisiae. pyogenesCas9 (wild type) (SEQ ID NO: 848), S. pyogenesCas9 mutant M1C (SEQ ID NO: 849), S. cerevisiae. pyogenesCas9-mutant M1C & C80S (SEQ ID NO: 850), S. pyogenesCas9 nickase mutant D10A (SEQ ID NO: 851), S. pylori. pyogenesCas9 nickase mutant H840A (SEQ ID NO: 852), S. pyogenesCas9 nickase mutants E923P & T924P (SEQ ID NO: 853), Acidovorax ebreus (Acidovorax ebreus) Cas9 (SEQ ID NO: 854), acid mine drainage fungus Ga0052161_JGI · Cas9 (SEQ ID NO: 855), at least about 75%, at least about 80%, at least about 85%, at least about 90% relative to a sequence selected from pyogenesCas9 null mutant D10A & H840A (SEQ ID NO: 1027) and uranium mine fungus FW106_JGI · Cas9 (sequence 856) Or a Cas9 protein having at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99 percent, or 100 percent amino acid sequence identity.
95. 95. The composition according to any one of embodiments 67 to 94, wherein the endosome escape agent comprises a lysosome agonist, a cell-penetrating peptide, a fusogenic peptide, a pore-forming agent, and a proton sponge agent. Said composition selected from.
96. 96. The composition of embodiment 95, wherein the endosomal escape agent is peptide ppTG21: GLFHALLHLLHSLWHLLLLHA (SEQ ID NO: 1012).
97. Embodiment 96. A pharmaceutical composition comprising the composition according to any one of embodiments 65 to 96 and a pharmaceutically acceptable carrier, excipient or diluent.
98. 98. A method of treating a disease or condition in a subject, comprising administering to said subject a therapeutically effective amount of a pharmaceutical composition according to embodiment 97.
99. 99. The method of embodiment 98, wherein the disease or condition is a blood disorder, cellular dysregulation or tumor disease and disorder, inflammation and immune related diseases, metabolic, liver, kidney, and protein diseases and disorders, Said method selected from the group consisting of muscular or skeletal diseases, neurological and neurological diseases and disorders, and eye diseases and disorders.
100. 99. A method of selectively modulating transcription of an intracellular target DNA of a subject, comprising administering to said subject the pharmaceutical composition of embodiment 97.
101. 101. The method of embodiment 100, wherein the target DNA is a blood disorder, cellular dysregulation or tumor disease and disorder, inflammation and immune related diseases, metabolic, liver, kidney, and protein diseases and disorders, muscle Or said method associated with a disorder or condition selected from the group consisting of skeletal diseases, neurological and neurological diseases and disorders, and eye diseases and disorders.
102. 98. A method of editing a nucleic acid molecule encoding a protein associated with a disease or condition in a subject, comprising administering to the subject the pharmaceutical composition of claim 97.
103. 102. The method of embodiment 102, wherein the protein is a blood disorder, cellular dysregulation or tumor disease and disorder, inflammation and immune related diseases, metabolic, liver, kidney, and protein diseases and disorders, muscle or Said method associated with a disease or condition selected from the group consisting of skeletal diseases, neurological and neurological diseases and disorders, and ocular diseases and disorders.
104. 98. A method of modulating the expression level of at least one gene product associated with a disease or condition of a subject, comprising administering to said subject the pharmaceutical composition of embodiment 97.
105. 105. The method of embodiment 104, wherein the gene product is a blood disorder, cellular dysregulation or tumor disease and disorder, inflammation and immune related diseases, metabolic, liver, kidney, and protein diseases and disorders, muscle. Or said method associated with a disease or condition selected from the group consisting of skeletal diseases, neurological and neurological disease disorders, and eye diseases and disorders.

Claims (29)

Compound of formula (A-1), (A-2) or (A-3):
Or a pharmaceutically acceptable salt thereof,
Where
R ^xx is, -H, - alkyl, - cycloalkyl, - alkenyl, - alkynyl, - aryl, - ^{heteroaryl, -OR 5, -N (R 4} ) -R 5, a -SR ^5, wherein R ^xx Each of —CH ₂ — groups may be independently replaced with a heteroatom group selected from —O—, —S—, —N (R ⁴ ) —, wherein —CH ₃ of R ^xx is , —N (R ⁴ ) ₂ , —OR ⁴ , and —S (R ⁴ ), which may be replaced with a heteroatom group, the heteroatom group being separated by at least two carbon atoms , The alkyl, alkenyl, alkynyl, and cycloalkyl each may be substituted with one or more halo atoms,
R ^yy represents —CN, —CH ₂ —CN, —C≡CH, —CH ₂ —N ₃ , —CH ₂ —NH ₂ , —CH ₂ —N (R ⁴ ) —S (O) ₂ —R ^5. , —CH ₂ —CO ₂ H, —CO ₂ H, —CH ₂ —OH, —CH ₂ —SH, —CH═CH—R ⁵ , —CH ₂ —R ⁵ , —CH ₂ —S—R ⁵ , ^{_{-CH 2 -N (R 4) -R}} 5, -CH 2 -N (R 4) -C (O) -R 5, -CH 2 -N (R 4) -C (O) -O-R 5 , —CH ₂ —N (R ⁴ ) —C (O) —N (R ⁴ ) —R ⁵ , —CH ₂ —O—R ⁵ , —CH ₂ —O—C (O) —R ⁵ , —CH ₂ —O—C (O) —N (R ⁴ ) —R ⁵ , —CH ₂ —O—C (O) —O—R ⁵ , —CH ₂ —S (O) —R ⁵ , —CH ₂ — ^{_{S (O) 2 -R 5,}} -CH 2 -S (O) 2 -N (R 4) -R 5, -C (O) -NH 2, -C (O) -O-R 5, -C (O -N (R ⁴⁾ -R ⁵ or aryl or heteroaryl, wherein aryl or heteroaryl are optionally substituted with R ^5,
Each R ¹ is independently —CN, —CH ₂ —CN, —C≡CH, —CH ₂ —N ₃ , —CH ₂ —NH ₂ , —CH ₂ —N (R ⁴ ) —S (O ) ₂ —R ⁵ , —CH ₂ —CO ₂ H, —CO ₂ H, —CH ₂ —OH, —CH ₂ —SH, —CH═CH—R ⁵ , —CH ₂ —R ⁵ , —CH ₂ — S—R ⁵ , —CH ₂ —N (R ⁴ ) —R ⁵ , —CH ₂ —N (R ⁴ ) —C (O) —R ⁵ , —CH ₂ —N (R ⁴ ) —C (O) —O—R ⁵ , —CH ₂ —N (R ⁴ ) —C (O) —N (R ⁴ ) —R ⁵ , —CH ₂ —O—R ⁵ , —CH ₂ —O—C (O) — R ⁵ , —CH ₂ —O—C (O) —N (R ⁴ ) —R ⁵ , —CH ₂ —O—C (O) —O—R ⁵ , —CH ₂ —S (O) —R ⁵ , —CH ₂ —S (O) ₂ —R ⁵ , —CH ₂ —S (O) ₂ —N (R ⁴ ) —R ⁵ , —C (O) —NH ₂ , —C (O) —O— R ⁵ Or, -C (O) -N (R 4) a -R ^5, or an aryl or heteroaryl, said aryl or heteroaryl is substituted with R ⁵ optionally,
Alternatively, R ¹ represents —Z—X—Y, —Z—Y, —X—Y, —Z—X ⁺ Y ⁻ , —X ⁺ Y ⁻ , —Z—X ⁻ Y ⁺ , —X ⁻ Y ^+. Or -Y,
X is a linker;
X ⁺ is a positively charged linker;
X ⁻ is a negatively charged linker,
Y is a ribonucleoprotein or endonuclease containing a site-specific modified polypeptide, Y is a site-specific modified polypeptide, Y is a Cas9 ribonucleoprotein, or Y is a Cas9 protein Or Y is a single guide RNA sequence (sgRNA) or Y is a dual guide RNA sequence comprising CRISPR RNA (crRNA) and transactive crRNA (tracrRNA);
Is Y ⁺ a positively charged ribonucleoprotein or endonuclease containing a site-specific modified polypeptide, Y ⁺ is a positively charged site-specific modified polypeptide, or Y ⁺ is positively A charged Cas9 protein,
Y ⁻ is a negatively charged ribonucleoprotein or endonuclease containing a site-specific modified polypeptide, Y ⁻ is a negatively charged site-specific modified polypeptide, or Y ⁻ is negative Either a charged Cas9 ribonucleoprotein, Y ⁻ is a negatively charged sgRNA, or Y ⁻ is a negatively charged dual guide RNA sequence comprising CRISPR RNA (crRNA) and transactive crRNA (tracrRNA) And
Z does not exist or is —C≡C—, —CH═CH—, —CH ₂ —, —CH ₂ —O—, —C (O) —N (R ⁴ ) —, —CH ₂ —. _{S -, - CH 2 -S (} O) -, - CH 2 -S (O) 2 -, - CH 2 -S (O) 2 -N (R 4) -, - C (O) -O-, ^{_{-CH 2 -N (R 4) -}} , - CH 2- N (R 4) -C (O) -, - CH 2 -N (R 4) -S (O) 2 -, - CH 2 -N ( R ⁴ ) —C (O) —O—, —CH ₂ —N (R ⁴ ) —C (O) —N (R ⁴ ) —, —CH ₂ —O—C (O) —, —CH ₂ — O—C (O) —N (R ⁴ ) —, —CH ₂ —O—C (O) —O—, or aryl or heteroaryl, wherein said aryl or heteroaryl is optionally substituted with R ⁵ ,
R ² represents —OH, —N ₃ , —N (R ³ ) ₂ , —N (R ³ ) —C (O) —R ³ , —N (R ³ ) —C (O) —N (R ³ ) ₂ , —N (R ³ ) —C (O) —OR ³ , —N (R ³ ) —S (O) ₂ —R ³ , tetrazole, or triazole, wherein the tetrazole and triazole are optionally R Replaced by ³ ,
Each R ³ is independently —H, — (C ₁ -C ₅ ) alkyl, halo-substituted (C ₁ -C ₅ ) alkyl, halo-substituted (C ₃ -C ₆ ) cycloalkyl, — (C _1- C ₅₎ alkenyl, - (C ₁ -C ₅₎ alkynyl, halo-substituted - (C ₁ -C ₅₎ alkenyl, halo-substituted - (C ₁ -C ₅₎ alkynyl, or (at C ₃ -C ₆₎ cycloalkyl And the —CH ₂ — group of said alkyl or cycloalkyl may each independently be replaced with a heteroatom group selected from —O—, —S—, and —N (R ⁴ ) —, The alkyl —CH ₃ may be independently replaced with a heteroatom group selected from —N (R ⁴ ) ₂ , —OR ⁴ , and —S (R ⁴ ). Are separated by at least two carbon atoms;
Each R ⁴ is independently —H, — (C ₁ -C ₂₀ ) alkyl, — (C ₁ -C ₂₀ ) alkenyl, — (C ₁ -C ₂₀ ) alkynyl, or (C ₃ -C ₆ ). 1-6 —CH ₂ — groups of said alkyl or cycloalkyl that are cycloalkyl and separated by at least 2 carbon atoms are each independently —O—, —S—, or —N (R ⁴⁾ - may be replaced with independently selected heteroatoms from, -CH ₃ of said alkyl, each independently, -N (R ⁴⁾ _2, -OR ^4, and -S (R ⁴ Wherein the heteroatom group is separated by at least two carbon atoms, and the alkyl, alkenyl, alkynyl, and cycloalkyl are substituted with a halo atom. Well,
Each R ⁵ is independently —H, (C ₃ -C ₂₀ ) cycloalkyl, — (C ₁ -C ₆₀ ) alkenyl, — (C ₁ -C ₆₀ ) alkynyl, or (C ₁ -C ₆₀ ). Alkyl, 1-6 —CH ₂ — groups of the cycloalkyl or 1-20 —CH ₂ — groups of the alkyl are each independently —O—, —S—, and —N; (R ⁴ ) — may be replaced with a heteroatom independently selected from wherein the heteroatom is separated by at least two carbon atoms, and the —CH ₃ of the alkyl is each independently May be exchanged with a heteroatom group selected from —N (R ⁴ ) ₂ , —OR ⁴ , and —S (R ⁴ ), said heteroatom group being separated by at least two carbon atoms; The alkyl, alkenyl, alkynyl, and cycloalkyl are substituted with a halo atom. It may be, the compound or a pharmaceutically acceptable salt thereof. Compound of formula (B):
Or a pharmaceutically acceptable salt thereof,
Where
R ¹ is, -Z-X-Y, -Z -Y, -X-Y, -Z-X + Y -, -X + Y -, -Z-X - Y +, -X - Y +, or -Y,
X is a linker;
X ⁺ is a positively charged linker;
X ⁻ is a negatively charged linker,
Y is a ribonucleoprotein or endonuclease containing a site-specific modified polypeptide, Y is a site-specific modified polypeptide, Y is a Cas9 ribonucleoprotein, or Y is a Cas9 protein Or Y is a single guide RNA sequence (sgRNA) or Y is a dual guide RNA sequence comprising CRISPR RNA (crRNA) and transactive crRNA (tracrRNA);
Is Y ⁺ a positively charged ribonucleoprotein or endonuclease containing a site-specific modified polypeptide, Y ⁺ is a positively charged site-specific modified polypeptide, or Y ⁺ is positively A charged Cas9 protein,
Y ⁻ is a negatively charged ribonucleoprotein or endonuclease containing a site-specific modified polypeptide, Y ⁻ is a negatively charged site-specific modified polypeptide, or Y ⁻ is negative Either a charged Cas9 ribonucleoprotein, Y ⁻ is a negatively charged sgRNA, or Y ⁻ is a negatively charged dual guide RNA sequence comprising CRISPR RNA (crRNA) and transactive crRNA (tracrRNA) And
Z does not exist or is —C≡C—, —CH═CH—, —CH ₂ —, —CH ₂ —O—, —C (O) —N (R ⁴ ) —, —CH ₂ —. _{S -, - CH 2 -S (} O) -, - CH 2 -S (O) 2 -, - CH 2 -S (O) 2 -N (R 4) -, - C (O) -O-, ^{_{-CH 2 -N (R 4) -}} , - CH 2- N (R 4) -C (O) -, - CH 2 -N (R 4) -S (O) 2 -, - CH 2 -N ( R ⁴ ) —C (O) —O—, —CH ₂ —N (R ⁴ ) —C (O) —N (R ⁴ ) —, —CH ₂ —O—C (O) —, —CH ₂ — O—C (O) —N (R ⁴ ) —, —CH ₂ —O—C (O) —O—, or aryl or heteroaryl, wherein said aryl or heteroaryl is optionally substituted with R ⁵ ,
R ² represents —OH, —N ₃ , —N (R ³ ) ₂ , —N (R ³ ) —C (O) —R ³ , —N (R ³ ) —C (O) —N (R ³ ) ₂ , —N (R ³ ) —C (O) —OR ³ , —N (R ³ ) —S (O) ₂ —R ³ , tetrazole, or triazole, wherein the tetrazole and triazole are optionally R Replaced by ³ ,
Each R ³ is independently —H, — (C ₁ -C ₅ ) alkyl, halo-substituted (C ₁ -C ₅ ) alkyl, halo-substituted (C ₃ -C ₆ ) cycloalkyl, — (C _1- C ₅₎ alkenyl, - (C ₁ -C ₅₎ alkynyl, halo-substituted - (C ₁ -C ₅₎ alkenyl, halo-substituted - (C ₁ -C ₅₎ alkynyl, or (at C ₃ -C ₆₎ cycloalkyl And the —CH ₂ — group of said alkyl or cycloalkyl may each independently be replaced with a heteroatom group selected from —O—, —S—, and —N (R ⁴ ) —, The —CH ₃ of the alkyl is independently —N (R ⁴ ) ₂ or —OR ^4.
And a heteroatom group selected from —S (R ⁴ ), wherein the heteroatom group is separated by at least two carbon atoms;
Each R ⁴ is independently —H, — (C ₁ -C ₂₀ ) alkyl, — (C ₁ -C ₂₀ ) alkenyl, — (C ₁ -C ₂₀ ) alkynyl, or (C ₃ -C ₆ ). 1-6 —CH ₂ — groups of said alkyl or cycloalkyl that are cycloalkyl and separated by at least 2 carbon atoms are each independently —O—, —S—, or —N (R ⁴⁾ - may be replaced with independently selected heteroatoms from, -CH ₃ of said alkyl, each independently, -N (R ⁴⁾ _2, -OR ^4, and -S (R ⁴ Wherein the heteroatom group is separated by at least two carbon atoms, and the alkyl, alkenyl, alkynyl, and cycloalkyl are substituted with a halo atom. Well,
Each R ⁵ is independently —H, (C ₃ -C ₂₀ ) cycloalkyl, — (C ₁ -C ₆₀ ) alkenyl, — (C ₁ -C ₆₀ ) alkynyl, or (C ₁ -C ₆₀ ). Alkyl, 1-6 —CH ₂ — groups of the cycloalkyl or 1-20 —CH ₂ — groups of the alkyl are each independently —O—, —S—, and —N; (R ⁴ ) — may be replaced with a heteroatom independently selected from wherein the heteroatom is separated by at least two carbon atoms, and the —CH ₃ of the alkyl is each independently May be exchanged with a heteroatom group selected from —N (R ⁴ ) ₂ , —OR ⁴ , and —S (R ⁴ ), said heteroatom group being separated by at least two carbon atoms; The alkyl, alkenyl, alkynyl, and cycloalkyl are substituted with a halo atom. It may be, the compound or a pharmaceutically acceptable salt thereof. The compound according to claim 1, wherein R ² is —NH—C (O) —CH ₃ . The compound according to claim 1, wherein R ¹ is —Z—X—Y. R ¹ is L1 to L10:
A compound according to claim 4, which is -Z-X-Y selected from the group consisting of
Here, each T is independently absent, or is (C ₁ -C ₁₀ ) alkylene, (C ₂ -C ₁₀ ) alkenylene, or (C ₂ -C ₁₀ ) alkynylene, Two or more carbon groups may each independently be replaced with a heteroatom group independently selected from —O—, —S—, and —N (R ⁴ ) —, Separated by at least two carbon atoms, wherein the alkylene, alkenylene, and alkynylene may each independently be substituted with one or more halo atoms;
Each Q is independently absent or C (O), C (O) —NR ⁴ , NR ⁴ —C (O), O—C (O) —NR ⁴ , NR ⁴ —C (O ) —O, —CH ₂ —, heteroaryl, or a heteroatom group selected from O, S, S—S, S (O), S (O) ₂ , and NR ⁴ , and at least two carbon atoms Separates the heteroatom group O, S, S—S, S (O), S (O) ₂ , and NR ⁴ from any other heteroatom group;
Each R ⁴ is independently —H, — (C ₁ -C ₂₀ ) alkyl, or (C ₃ -C ₆ ) cycloalkyl, wherein the alkyl or cycloalkyl is separated by at least two carbon atoms. 1 to 6 —CH ₂ — groups may each independently be replaced with —O—, —S—, or —N (R ⁴ ) —, and —CH ₃ of the alkyl is —N ^{(R 4) 2, -OR 4} , and may be replaced with a heteroatom group selected from -S (R ^4), wherein the hetero atom group is spaced by at least two carbon atoms, said alkyl And cycloalkyl may be substituted with a halo atom,
Each m is independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, Said compound which is 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40.   6. A compound according to claim 5, wherein X comprises a disulfide bond. Formula (C-1), (C-2), (C-3), or (C4):
A compound according to claim 1 having the formula: or a pharmaceutically acceptable salt thereof,
Where
n is 1, 2 or 3,
W is absent or is a peptide,
L is − (TQTQ) _m ,
Each T is independently absent or (C ₁ -C ₁₀ ) alkylene, (C ₂ -C ₁₀ ) alkenylene, or (C ₂ -C ₁₀ ) alkynylene, wherein one or more of said T Tansomoto are each independently, -O -, - S-, and -N (R ⁴⁾ - may be replaced with a heteroatom groups independently selected from the hetero atom group is at least 2 Are separated by one carbon atom, wherein the alkylene, alkenylene, and alkynylene may each independently be substituted with one or more halo atoms;
Each Q is independently absent or C (O), C (O) —NR ⁴ , NR ⁴ —C (O), O—C (O) —NR ⁴ , NR ⁴ —C (O ) —O, —CH ₂ —, heteroaryl, or a heteroatom group selected from O, S, S—S, S (O), S (O) ₂ , and NR ⁴ , and at least two carbon atoms Separates the heteroatom group O, S, S—S, S (O), S (O) ₂ , and NR ⁴ from any other heteroatom group;
Each R ⁴ is independently —H, — (C ₁ -C ₂₀ ) alkyl, or (C ₃ -C ₆ ) cycloalkyl, wherein the alkyl or cycloalkyl is separated by at least two carbon atoms. 1 to 6 —CH ₂ — groups may be replaced with —O—, —S—, or —N (R ⁴ ) —, and each —CH ₃ of the alkyl is independently —N _{^{(R 4) 2, -OR 4}} , and may be replaced with a heteroatom group selected from -S (R ^4), wherein the hetero atom group is spaced by at least two carbon atoms, said alkyl And cycloalkyl may be substituted with a halo atom,
Each m is independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 Acceptable salt. Formula (E):
A compound according to claim 2 having the formula: or a pharmaceutically acceptable salt thereof,
Where
n is 1, 2 or 3,
W is absent or is a peptide,
L is − (TQTQ) _m ,
Each T is independently absent, or (C ₁ -C ₁₀₎ alkylene, (C ₂ -C ₁₀₎ alkenylene, or (C ₂ -C ₁₀₎ alkynylene, one or more of the T Tansomoto are each independently, -O -, - S-, and -N (R ⁴⁾ - may be replaced with a heteroatom groups independently selected from the hetero atom group is at least 2 Separated by two carbon atoms,
Each Q is independently absent or C (O), C (O) —NR ⁴ , NR ⁴ —C (O), O—C (O) —NR ⁴ , NR ⁴ —C (O ) —O, —CH ₂ —, heteroaryl, or a heteroatom group selected from O, S, S—S, S (O), S (O) ₂ , and NR ⁴ , and at least two carbon atoms Separates the heteroatom group O, S, S—S, S (O), S (O) ₂ , and NR ⁴ from any other heteroatom group;
Each R ⁴ is independently —H, — (C ₁ -C ₂₀ ) alkyl, or (C ₃ -C ₆ ) cycloalkyl, wherein the alkyl or cycloalkyl is separated by at least two carbon atoms. 1 to 6 —CH ₂ — groups may each independently be replaced with —O—, —S—, or —N (R ⁴ ) —, and —CH ₃ of the alkyl is —N Optionally substituted with a heteroatom group selected from (R ⁴ ) ₂ , —OR ⁴ , and —S (R ⁴ ), wherein the heteroatom group is separated by at least two carbon atoms and the alkyl And cycloalkyl may be substituted with a halo atom, and each m is independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 2 , 29,30,31,32,33,34,35,36,37,38,39 or 40, wherein the compound or a pharmaceutically acceptable salt thereof. The compound has the formula
The compound according to claim 8 or a pharmaceutically acceptable salt thereof, wherein n ′ is 1 or 2.   Y is a Cas9 ribonucleoprotein, which is a recognition element comprising either (1) a dual guide RNA sequence including CRISPR RNA (crRNA) and transactive crRNA (tracrRNA), or a single guide RNA sequence (sgRNA) Wherein the guide sequence directs sequence-specific binding of the Cas9 ribonucleoprotein to a target sequence upon expression, and optionally comprises one or more endosomal escape agents. One element, and (2) a second element comprising a Cas9 protein and optionally one or more nuclear localization sequences (NLS) and optionally one or more fluorescent proteins, and one or more endosomal escape agents 3. The compound of claim 1 or 2, wherein the first element is associated with the second element.   11. The compound of claim 10, wherein Y comprises one or two NLS, each of said NLS comprising the amino acid sequence PKKKRRKV (SEQ ID NO: 830).   Y is S.I. aureus, S .; pneumoniae, S .; pyogenes, S .; thermophilus, N.M. meningitidis, or A. at least about 75 percent, at least about 80 percent, at least about 85 percent, at least about 90 percent, at least about 95 percent, at least about 96 percent, at least about 97 percent, at least about 98 percent, at least about Cas9 protein from ebreus 3. The compound of claim 1 or 2, comprising a Cas9 protein that is about 99 percent, or 100 percent amino acid sequence identity.   Y is S.I. pyogenes Cas9 (wild type) (SEQ ID NO: 848), S. pyogenes Cas9 mutant M1C (SEQ ID NO: 849), S. cerevisiae. pyogenes Cas9 mutants M1C and C80S (SEQ ID NO: 850), S. cerevisiae. pyogenes Cas9 nickase mutant D10A (SEQ ID NO: 851), S. cerevisiae. pyogenes Cas9 nickase mutant H840A (SEQ ID NO: 852), S. pyogenes Cas9 nickase variants E923P and T924P (SEQ ID NO: 853), Acidovorax ebreus Cas9 (SEQ ID NO: 854), acidic mine drainage bacteria Ga0052161_JGI Cas9 (SEQ ID NO: 855), S. at least about 75 percent, at least about 80 percent, at least about 85 percent, at least about a sequence selected from pyogenes Cas9 null mutants D10A and H840A (SEQ ID NO: 1027), and the uranium mine bacterium FW106_JGI Cas9 (SEQ ID NO: 856) 3. The Cas9 protein comprising about 90 percent, at least about 95 percent, at least about 96 percent, at least about 97 percent, at least about 98 percent, at least about 99 percent, or 100 percent amino acid sequence identity. The described compound.   The compound according to claim 1 or 2, wherein the compound further comprises an endosome escape agent.   15. The compound of claim 14, wherein the endosomal escape agent is peptide ppTG21: GLFHALLHLLHSLWHLLLLHA (SEQ ID NO: 1012).   3. A composition comprising the compound of claim 1 or 2 and an endosome escape agent, wherein the compound and the endosome escape agent are co-incubated to form the composition.   A pharmaceutical composition comprising a compound according to claim 1 or 2 and a pharmaceutically acceptable carrier, excipient or diluent.   18. A method of treating a liver disease or condition or liver modulation disease or condition in a subject, comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition according to claim 17.   The disease or condition is hereditary angioedema, familial tyrosinemia type I, Alagille syndrome, alpha-1 antitrypsin deficiency, bile acid synthesis and metabolism abnormality, biliary atresia, cystic fibrosis liver disease, idiopathic Neonatal hepatitis, mitochondrial hepatic disorder, progressive familial intrahepatic cholestasis, primary sclerosing cholangitis, transthyretin amyloidosis, hemophilia, homozygous familial hypercholesterolemia, familial chylomicronemia , Hyperlipidemia, steatohepatitis, nonalcoholic steatohepatitis (NASH), nonalcoholic fatty liver disease (NAFLD), hyperglycemia, and diseases with abnormally high liver glucose production similar to type II diabetes The method of claim 18, wherein the method is selected from the group consisting of:   18. A method for selectively modulating transcription of a target DNA in a subject's liver cells, comprising administering to the subject a pharmaceutical composition according to claim 17.   17. A method of editing a nucleic acid molecule that encodes a protein associated with a liver disease or condition in a subject, the method comprising administering to the subject a pharmaceutical composition according to claim 16.   18. A method for modulating the expression of a level of at least one gene product associated with a liver disease or condition in a subject, comprising administering to the subject a pharmaceutical composition according to claim 17.   A composition comprising a ribonucleoprotein and an endosome escape agent.   24. The composition of claim 23, wherein the ribonucleoprotein is a Cas9 ribonucleoprotein or a Cpf1 ribonucleoprotein.   24. A pharmaceutical composition comprising the composition of claim 23 and a pharmaceutically acceptable carrier, excipient or diluent.   26. A method of treating a disease or condition in a subject comprising administering to said subject a therapeutically effective amount of the pharmaceutical composition of claim 25.   26. A method for selectively modulating transcription of target DNA in a cell of a subject comprising administering to said subject a pharmaceutical composition according to claim 25.   26. A method of editing a nucleic acid molecule encoding a protein associated with a disease or condition in a subject, comprising administering to the subject a pharmaceutical composition according to claim 25.   26. A method of modulating the expression of a level of at least one gene product associated with a disease or condition in a subject, comprising administering to the subject a pharmaceutical composition according to claim 25.